(12) Patent Application Publication (10) Pub. No.: US 2017/0198308A1 QI Et Al

Home , Apelin receptor, Bradykinin receptor B1, Caspase 12, Caspase 7, Clostripain, Dopamine receptor D2

US 20170 1983O8A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0198308A1 QI et al. (43) Pub. Date: Jul. 13, 2017

(54) CHMERIC PROTEINS AND METHODS OF filed on Jun. 17, 2016, provisional application No. REGULATING GENE EXPRESSION 62/399,902, filed on Sep. 26, 2016.

(71) Applicant: THE BOARD OF TRUSTEES OF Publication Classification THE LELAND STANFORD JUNOR UNIVERSITY, Stanford, CA (US) (51) Int. C. CI2N 15/90 (2006.01) (72) Inventors: LEI S. QI, Stanford, CA (US): P.C. CI2N 9/22 (2006.01) DAVE P. DINGAL, Stanford, CA (US) (52) U.S. C. CPC ...... CI2N 15/907 (2013.01); C12N 9/22 (73) Assignee: THE BOARD OF TRUSTEES OF (2013.01); C12Y-301/00 (2013.01) THE LELAND STANFORD JUNOR UNIVERSITY, Stanford, CA (US) (57) ABSTRACT (21) Appl. No.: 15/403,058 The present disclosure provides systems, compositions and methods for regulating expression of a target polynucleotide (22) Filed: Jan. 10, 2017 in a cell. The systems, compositions and methods comprise a chimeric receptor polypeptide comprising a G-protein Related U.S. Application Data coupled receptor (GPCR) or a fragment thereof, a chimeric (60) Provisional application No. 62/277.322, filed on Jan. adaptor polypeptide, at least one actuator moiety and a 11, 2016, provisional application No. 62/351,522, cleavage moiety. Patent Application Publication Jul. 13, 2017 Sheet 1 of 49 US 2017/O1983O8A1

FIGURE I

102

FIGURE 2

extracellular

intracellular

2O3 Patent Application Publication Jul. 13, 2017 Sheet 2 of 49 US 2017/O1983O8A1

FIGURE 3A

3OOb

FIGURE 3B

301 -

303 302 302a - N 3O4. Patent Application Publication Jul. 13, 2017. Sheet 3 of 49 US 2017/O1983O8A1

.*(~~~~)....…*********

!**,.ae***) ----*******~. Patent Application Publication Jul. 13, 2017 Sheet 4 of 49 US 2017/O1983O8A1

FIGURES

extracellular

intracellular Patent Application Publication Jul. 13, 2017 Sheet 5 of 49 US 2017/O1983O8A1

FIGURE 6A

FIGURE 6B

6OOb Patent Application Publication Jul. 13, 2017 Sheet 6 of 49 US 2017/O1983O8A1

FIGURE 7

705 Patent Application Publication Jul. 13, 2017. Sheet 7 of 49 US 2017/O1983O8A1

Patent Application Publication Jul. 13, 2017 Sheet 8 of 49 US 2017/O1983O8A1

FIGURE 9

907 Patent Application Publication Jul. 13, 2017. Sheet 9 of 49 US 2017/O1983O8A1

(10IGIRIOEDI-H?IH80GI?+{101,

….….!!!!!--****... (H0][[{{{ISOIH Patent Application Publication Jul. 13, 2017. Sheet 10 of 49 US 2017/0198308A1

FIGURE 11

Patent Application Publication Jul. 13, 2017. Sheet 11 of 49 US 2017/0198308A1

FIGURE 2A FIGURE 12B FIGURE 2C

2OO Patent Application Publication Jul. 13, 2017. Sheet 12 of 49 US 2017/O1983O8A1

§§§§§§

V?I(HYHÍTROIH Patent Application Publication Jul. 13, 2017. Sheet 13 of 49 US 2017/O1983O8A1

****

::::::::::

vritanoia Patent Application Publication Jul. 13, 2017. Sheet 14 of 49 US 2017/O1983O8A1

FIGURE 15

88:38:33.

3.x.

3.a.

8.8

iv putative) Patent Application Publication Jul. 13, 2017. Sheet 15 of 49 US 2017/O1983O8A1

|O9LGIRHOEDI-H ~…………” · |×~ (sevesose)€809 6euenoore):) Patent Application Publication Jul. 13, 2017. Sheet 16 of 49 US 2017/0198308A1

Patent Application Publication Jul. 13, 2017. Sheet 17 of 49 US 2017/O1983O8A1

Patent Application Publication Jul. 13, 2017. Sheet 18 of 49 US 2017/0198308A1

FIGURE 9A FIGURE 9B FIGURE 9C

: 8:8; 8x8 8:g:: $:::::::::::::::::888 28 sig 8 is

FIGURE 9) FIGURE9E FIGURE 9F

3:338:::::::::::::::::::::8 ::::::::::::::::s: $88:88. *::::::::::::::::::::::::::::388 k i. Patent Application Publication Jul. 13, 2017. Sheet 19 of 49 US 2017/O1983O8A1

&####### Patent Application Publication Jul. 13, 2017. Sheet 20 of 49 US 2017/0198308A1

FIGURE 2A

2 N {yari: 88&e:

88:883 tecoratiris: chier8sic 388.883:33

88: 388 iss

38:3:88s 25

888x888 kixes: - s: $88 ::::::::: ::ce a xx-x- Patent Application Publication Jul. 13, 2017. Sheet 21 of 49 US 2017/0198308A1

FIGURE 21B

216 Patent Application Publication Jul. 13, 2017. Sheet 22 of 49 US 2017/0198308A1

FIGURE 22A

22 2.

::::::::: & 8 Q s:*w-g::::::ty-888 Y 22

FIGURE 22B

y is 3rd -- *ixios:cti, 3. Coiage

mCherry Patent Application Publication Jul. 13, 2017. Sheet 23 of 49 US 2017/0198308A1

FIGURE 22C FIGURE 22 D

3. regarter intersity a.i.

-8 $38,8883: 3888:38t {&is £883

FIGURE 22E A. A

mCherry

GFP Patent Application Publication Jul. 13, 2017. Sheet 24 of 49 US 2017/0198308A1

Patent Application Publication Jul. 13, 2017. Sheet 25 of 49 US 2017/0198308A1

FIGURE 23B

&:S383.38: 8:::

3. 8:888&3.88.

Patent Application Publication Jul. 13, 2017. Sheet 26 of 49 US 2017/0198308A1

FIGURE 3C 3.

FIGURE 3D

F. reporter intensity 3.E.

A-8 8 A+ A- &- A+ r; 3-A, S: --S {^{i} {{C} Patent Application Publication Jul. 13, 2017. Sheet 27 of 49 US 2017/0198308A1

FIGURE 23E

FGURE 23F

; : 8 : a 8 8. SFF g reporter 3 : s s intersity is 8 a.is...} :

SF reporter intensity 8,8. C: 3-C88C CKO --C8 Patent Application Publication Jul. 13, 2017. Sheet 28 of 49 US 2017/O1983O8A1

& 8888&s:

§§*****

338: 0.3838: it

vºzannoia Patent Application Publication Jul. 13, 2017. Sheet 29 of 49 US 2017/0198308A1

FIGURE 25A FIGURE 2SB FIGURE 25C

Patent Application Publication Jul. 13, 2017. Sheet 30 of 49 US 2017/0198308A1

FIGURE 26A

8:3:3-38:3&tic ------ee-represerterer : Sk 3. 388 i& 38k. &S.

FIGURE 26B -eika

C3888

t ...... 28. S8& 8% S6K Wief. Patent Application Publication Jul. 13, 2017. Sheet 31 of 49 US 2017/0198308A1

FCURE 26C

CASP8 CCND CDKN1B Nothing FIGURE 26D 100 ss

CASP8 CCND1 CDKN1B Nothing Patent Application Publication Jul. 13, 2017. Sheet 32 of 49 US 2017/0198308A1

FIGURE 27A FIGURE 27B

i{} 8::::::: *:::::::ce:Kx 3.33. Patent Application Publication Jul. 13, 2017. Sheet 33 of 49 US 2017/0198308A1

FIGURE 28A

FIGURE 28B {{

3. {

{ o

CXCI2 ligand Concentration Patent Application Publication Jul. 13, 2017. Sheet 34 of 49 US 2017/0198308A1

ex e r s 3. g K {} k P ex Aouen be

Patent Application Publication Jul. 13, 2017. Sheet 35 of 49 US 2017/O1983O8A1

38%.*********& ?s&&&&& ******??; ¿? Patent Application Publication Jul. 13, 2017. Sheet 36 of 49 US 2017/0198308A1

FIGURE 31 A INPUT: Native Extraxeiia Sigirai

(it segrassaixie 3888 &g:iatic:

FIGURE 31B Development of chimeric Notch receptors

Patent Application Publication Jul. 13, 2017. Sheet 37 of 49 US 2017/0198308A1

FIGURE 31C FIGURE 3D

Deta-tepeciet Activatic

FIGURE 3E FIGURE 3F

& 3:. 88: 8x3:8: Rex:3f:2pg:RE 4x8 sites &x.8 es

Patent Application Publication Jul. 13, 2017. Sheet 38 of 49 US 2017/0198308A1

FGURE 3G FIGURE 3H EGFP Reporter Assay {} 88 {x. 8:8883-888 88: 8:8: ** 3:3: 388 inhibitor : 888 &: 8, 8:33, 8 :ex {3.3 ::::::::::::: s .. 3. 888 88xxter Activatics:

FIGURE 3

Patent Application Publication Jul. 13, 2017. Sheet 39 of 49 US 2017/0198308A1

FIGURE 32A FIGURE 32B

8:888 as: £8:3-& £8388.88%t

isig, 8: {&isci: &38sists 8:8:8 88s. 8x8 is

& 8388: 3. it is 3:8688 &888 38%. 38883: E833. 883:33 &&.8: 8x33x3: 88883:

FIGURE 32C

38888.

stroyerseer

six & Patent Application Publication Jul. 13, 2017. Sheet 40 of 49 US 2017/0198308A1

FIGURE 33

Patent Application Publication Jul. 13, 2017. Sheet 41 of 49 US 2017/0198308A1

FIGURE 34 xix-isixxxxix. 8xxxix.

Patent Application Publication Jul. 13, 2017. Sheet 42 of 49 US 2017/0198308A1

FIGURE 35A

FIGURE 35B

is 38 s ac 8 88: 8 8:88-8:8; 8.3.3 888;&E ::ite &xxxx Patent Application Publication Jul. 13, 2017. Sheet 43 of 49 US 2017/0198308A1

FIGURE 35C FIGURE 35D

C388-f 83&ies 838:38c

3.8%

is,

3. & : & W :::: 8 E3: *eporter itersity 3.8. Patent Application Publication Jul. 13, 2017. Sheet 44 of 49 US 2017/0198308A1

FIGURE 36A reta-tepeciet (serie Cutting &

FIGURE 36B

Patent Application Publication Jul. 13, 2017. Sheet 45 of 49 US 2017/0198308A1

FIGURE 36C

II. 388 : 3:38 8:8 --- &

FIGURE 36D

8:3& 3888

FIGURE 36E % (XR-3-8gative cesis 3. Patent Application Publication

Patent Application Publication Jul. 13, 2017. Sheet 47 of 49 US 2017/0198308A1

FIGURE 38

sgCXCR4Minimal NCS sgCD95variant l CXCR4 CD95

an. CD95 (a.u.) dCas).-VPR Minimal NCS variant

sgOXCR4 sgOD95 - Deits scxcR4 ii, ; : sg(XC: a sig8S Beita sgOXCR4 is sigCS3 - Deita is 8 sgx: sg38 keta ki Patent Application Publication Jul. 13, 2017. Sheet 48 of 49 US 2017/0198308A1

FIGURE 39A FIGURE 39B

i:igi:

s : s R sy ise-pre-t-cis--- k NA test CE. CYCLE

FIGURE 39C FIGURE 39D

83.88.* . . :::::::::::::*::: 8. & DNA content -- sg:KN; 8 Patent Application Publication Jul. 13, 2017. Sheet 49 of 49 US 2017/0198308A1

FIGURE 39E

4. DAP

: Nicotest ->

FIGURE 39F

4. DApr & DAP: US 2017/O 1983O8 A1 Jul. 13, 2017

CHMERIC PROTEINS AND METHODS OF cleavage recognition site; wherein: (i) the GMP forms a REGULATING GENE EXPRESSION portion of an intracellular region of the chimeric receptor polypeptide, and the cleavage moiety forms a portion of the CROSS-REFERENCE chimeric adaptor polypeptide; (ii) the GMP forms a portion 0001. This application claims the benefit of U.S. Provi of the chimeric adaptor polypeptide, and the cleavage moi sional Application No. 62/277.322 filed on Jan. 11, 2016, ety forms a portion of an intracellular region of the chimeric U.S. Provisional Application No. 62/351,522 filed on Jun. receptor polypeptide; or (iii) the cleavage moiety is com 17, 2016, and U.S. Provisional Application No. 62/399,902 plexed with a second adaptor polypeptide that binds the filed on Sep. 26, 2016, each of which is incorporated in its chimeric receptor polypeptide in response to the receptor modification, and the GMP forms a portion of the chimeric entirety herein by reference. adaptor polypeptide. In some embodiments, the receptor REFERENCE TO ASEQUENCE LISTING does not comprise SEQ ID NO:39. 0006. In some embodiments, the target polynucleotide is 0002. The Sequence Listing written in file 0794.45 genomic DNA. In some embodiments, the target polynucle 000830US-1015078 SequenceListing..txt, created on Mar. otide is RNA. In some embodiments, the modification is 8, 2017, 189,094 bytes, machine format IBM-PC, MS phosphorylation. Windows operating system, is hereby incorporated by ref 0007. In some embodiments, the actuator moiety is a Cas erence in its entirety for all purposes protein, and the system further comprises a guide RNA BACKGROUND active to form a complex with the Cas protein. In some embodiments, (i) the actuator moiety is an RNA binding 0003) Regulation of cell activities can involve the binding protein (RBP) optionally complexed with a guide RNA, and of a ligand to a membrane-bound receptor comprising an (ii) the system further comprises a Cas protein that is able to extracellular ligand binding domain and an intracellular form a complex with the guide RNA. In some embodiments, (e.g., cytoplasmic) signaling domain. The formation of a the Cas protein substantially lacks DNA cleavage activity. complex between a ligand and the ligand binding domain 0008. In some embodiments, (i) the GMP forms a portion can result in a conformational and/or chemical modification of the chimeric adaptor polypeptide, (ii) cleavage of the in the receptor which can result in a signal transduced within the cell. In some situations, the cytoplasmic portion of the cleavage recognition site is effective to release the chimeric receptor is phosphorylated (e.g., trans- and/or auto-phos adaptor polypeptide from the receptor, and (iii) the system phorylated), resulting in a change in its activity. These comprises a further chimeric adaptor polypeptide compris events can be coupled with secondary messengers and/or the ing an GMP that binds to the modified receptor. recruitment of co-factor proteins. In some instances, the 0009. In some embodiments, receptor modification com change in the cytoplasmic portion results in binding to other prises modification at multiple modification sites, and each proteins (e.g., co-factor proteins and/or other receptors). modification site is effective to bind an adaptor polypeptide. These other proteins can be activated and then carry out 0010. In some embodiments, the cleavage recognition various functions within a cell. site comprises a polypeptide sequence, and the cleavage 0004 Conditional gene expression systems allow for moiety comprises protease activity. In some embodiments, conditional regulation of one or more target genes. Condi the cleavage recognition site comprises a disulfide bond, and tional gene expression systems such as drug-inducible gene the cleavage moiety comprises oxidoreductase activity. In expression systems allow for the activation and/or deacti Some embodiments, the cleavage recognition site comprises Vation of gene expression in response to a stimulus, such as a first portion of an intein sequence that reacts with a second the presence of a drug. Currently available systems, how portion of the intein sequence to release the actuator moiety. ever, can be limited due to imprecise control, insufficient 0011. In some embodiments, the receptor is a transmem levels of induction (e.g., activation and/or deactivation of brane receptor. In some embodiments, the receptor is a gene expression), and lack of specificity. nuclear receptor. 0012. In some embodiments, the actuator moiety regu SUMMARY lates expression of the target polynucleotide by physical 0005. In view of the foregoing, there exists a consider obstruction of the target polynucleotide or recruitment of able need for alternative compositions and methods to carry additional factors effective to Suppress or enhance expres out conditional regulation of gene expression, for example sion of the target polynucleotide. In some embodiments, the by regulating expression of a target polynucleotide. In an actuator moiety comprises an activator effective to increase aspect, the present disclosure provides a system for regu expression of the target polynucleotide. In some embodi lating expression of a target polynucleotide in a cell. In some ments, the actuator moiety is linked to at least one nuclear embodiments, the system comprises (a) a chimeric receptor localization signal (NLS). polypeptide that is modified upon binding an antigen, 0013. In some embodiments, the chimeric receptor poly wherein receptor modification comprises a conformational peptide is linked to at least one targeting sequence which change or chemical modification; (b) a chimeric adaptor directs transport of the receptor to a specific region of a cell. polypeptide that binds the receptor in response to the recep In some embodiments, the targeting sequence directs trans tor modification; (c) a gene modulating polypeptide (GMP) port of the receptor to a nucleus, cytoplasm, mitochondria, comprising an actuator moiety linked to a cleavage recog endoplasmic reticulum (ER), chloroplast, apoplast, peroxi nition site, wherein upon cleavage of the cleavage recogni Some or plasma membrane. In some embodiments, the tion site, the actuator moiety is activated to complex with a targeting sequence comprises a nuclear export signal (NES). target polynucleotide; and (d) a cleavage moiety that cleaves In some embodiments, the targeting sequence comprises a the cleavage recognition site when in proximity to the plasma membrane targeting peptide. US 2017/O 1983O8 A1 Jul. 13, 2017

0014. In some embodiments, the chimeric adaptor poly moiety comprises protease activity. In some embodiments, peptide is linked to at least one targeting sequence which the cleavage recognition site comprises a disulfide bond, and directs transport of the adaptor to a specific region of a cell. the cleavage moiety comprises oxidoreductase activity. In In some embodiments, the targeting sequence directs trans Some embodiments, the cleavage recognition site comprises port of the chimeric adaptor polypeptide to a nucleus, a first portion of an intein sequence that reacts with a second cytoplasm, mitochondria, endoplasmic reticulum (ER). portion of the intein sequence to release the actuator moiety. chloroplast, apoplast, peroxisome or plasma membrane. In 0022. In some embodiments, the receptor is a transmem Some embodiments, the targeting sequence comprises a brane receptor. In some embodiments, the receptor is a nuclear export signal (NES). In some embodiments, the targeting sequence comprises a plasma membrane targeting nuclear receptor. peptide. 0023. In some embodiments, the actuator moiety regu 0015. In some embodiments, the receptor is linked to a lates expression of the target polynucleotide by physical polypeptide folding domain. In some embodiments, the obstruction of the target polynucleotide or recruitment of chimeric adaptor polypeptide is linked to a polypeptide additional factors effective to Suppress or enhance expres folding domain. sion of the target polynucleotide. In some embodiments, the 0016. In an aspect, the present disclosure provides a actuator moiety comprises an activator effective to increase method of regulating expression of a target polynucleotide expression of the target polynucleotide. in a cell. In some embodiments, the method comprises (a) 0024. In an aspect, the present disclosure provides a exposing a chimeric receptor polypeptide to an antigen, chimeric intracellular receptor. In some embodiments, the wherein (i) the receptor is modified upon exposure to the receptor comprises (a) an antigen interacting domain that antigen, and (ii) receptor modification comprises a confor specifically binds an antigen; and (b) an actuator moiety mational change or a chemical modification; (b) binding a linked to the antigen interacting domain; wherein: (i) the chimeric adaptor polypeptide to the chimeric receptor poly chimeric intracellular receptor is modified in response to peptide in response to receptor modification to form a antigen binding; (ii) the chimeric receptor polypeptide trans complex between a gene modulating polypeptide (GMP) locates to a nucleus of a cell in response to modification; and and a cleavage moiety, wherein the GMP comprises an (iii) the actuator moiety complexes with a target polynucle actuator moiety linked to a cleavage recognition site; and (c) otide in the nucleus. cleaving the cleavage recognition site with the cleavage 0025. In some embodiments, the actuator moiety is a Cas moiety, wherein upon cleavage of the cleavage recognition protein that forms a complex with a guide RNA. In some site, the actuator moiety complexes with a target polynucle embodiments, the Cas protein substantially lacks DNA otide thereby regulating expression of the target polynucle cleavage activity. otide in the cell; wherein: (i) the GMP forms a portion of an intracellular region of the chimeric receptor polypeptide, 0026. In some embodiments, the actuator moiety regu and the cleavage moiety forms portion of the chimeric lates expression of the target polynucleotide by physical adaptor polypeptide; (ii) the cleavage moiety forms part of obstruction of the target polynucleotide or recruitment of the chimeric adaptor polypeptide, and the GMP forms a additional factors effective to Suppress or enhance expres portion of an intracellular region of the chimeric receptor; or sion of the target polynucleotide. In some embodiments, the (iii) the cleavage moiety is complexed with a second adaptor actuator moiety comprises an activator effective to increase polypeptide that binds the receptor in response to the recep expression of the target polynucleotide. tor modification, and the GMP forms a portion of the 0027 In some embodiments, the antigen is a hormone. chimeric adaptor polypeptide. In some embodiments, the receptor does not comprise SEQ ID NO: 39. 0028. In some embodiments, the actuator moiety is 0017. In some embodiments, the target polynucleotide is linked to at least one nuclear localization signal (NLS). genomic DNA. In some embodiments, the target polynucle 0029. In some embodiments, the receptor is linked to at otide is RNA. In some embodiments, the modification is least one targeting sequence which directs transport of the phosphorylation. receptor to a specific region of a cell. In some embodiments, 0018. In some embodiments, the actuator moiety is a Cas the targeting sequence directs transport of the receptor to a protein that forms a complex with a guide RNA. In some nucleus, cytoplasm, mitochondria, endoplasmic reticulum embodiments, the actuator moiety is an RNA binding pro (ER), chloroplast, apoplast, or peroxisome. In some embodi tein (RBP) complexed with a guide RNA that forms a ments, the targeting sequence comprises a nuclear export complex with a Cas protein. In some embodiments, the Cas signal (NES). In some embodiments, the targeting sequence protein substantially lacks DNA cleavage activity. comprises plasma membrane targeting peptide. In some 0019. In some embodiments, (i) the GMP forms a portion embodiments, the receptor is linked to a polypeptide folding of the chimeric adaptor polypeptide, (ii) the chimeric adap domain. tor polypeptide is released from the receptor following 0030. In an aspect, the present disclosure provides a cleavage of the cleavage recognition site, and (iii) a further method of regulating expression of a target polynucleotide chimeric adaptor polypeptide comprising an GMP binds the in a cell comprising a nucleus. In some embodiments, the modified receptor. method comprises (a) exposing a chimeric intracellular 0020. In some embodiments, receptor modification com receptor to an antigen, wherein (i) the receptor comprises an prises modification at multiple modification sites, and each antigen interacting domain and actuator moiety, and (ii) the modification site is effective to bind a chimeric adaptor receptor is modified upon exposure to the antigen; (b) polypeptide. translocating the modified receptor to the nucleus; and (c) 0021. In some embodiments, the cleavage recognition forming a complex between the actuator moiety and the site comprises a polypeptide sequence, and the cleavage target polynucleotide. US 2017/O 1983O8 A1 Jul. 13, 2017

0031. In some embodiments, the actuator moiety is a Cas expression of the target polynucleotide. In some embodi protein that forms a complex with a guide RNA. In some ments, the actuator moiety is linked to at least one nuclear embodiments, the Cas protein substantially lacks DNA localization signal (NLS). cleavage activity. 0042. In some embodiments, the receptor is linked to at 0032. In some embodiments, the actuator moiety regu least one targeting sequence which directs transport of the lates expression of the target polynucleotide by physical receptor to a specific region of a cell. In some embodiments, obstruction of the target polynucleotide or recruitment of the targeting sequence directs transport of the receptor to a additional factors effective to Suppress or enhance expres nucleus, cytoplasm, mitochondria, endoplasmic reticulum sion of the target polynucleotide. In some embodiments, the (ER), chloroplast, apoplast, peroxisome or plasma mem actuator moiety comprises an activator effective to increase brane. In some embodiments, the targeting sequence com expression of the target polynucleotide. prises a nuclear export signal (NES). In some embodiments, 0033. In some embodiments, the antigen is a hormone. the targeting sequence comprises a plasma membrane tar 0034. In an aspect, the present disclosure provides a geting peptide. In some embodiments, the receptor is linked chimeric receptor polypeptide. In some embodiments, a to a polypeptide folding domain. chimeric receptor polypeptide comprises (a) an antigen 0043. In an aspect, the present disclosure provides a interacting domain ; and (b) a gene modulating polypeptide chimeric adaptor polypeptide. In some embodiments, a (GMP) comprising an actuator moiety linked to a cleavage chimeric adaptor polypeptide comprises (a) a receptor bind recognition site; wherein: (i) the chimeric receptor polypep ing moiety that binds a receptor that has undergone modi tide is modified in response to antigen binding; (ii) the fication upon binding to an antigen; and (b) a gene modu cleavage recognition site is cleaved by a cleavage moiety in lating polypeptide (GMP) linked to the receptor binding response to modification of the chimeric receptor polypep moiety, wherein the GMP comprises an actuator moiety tide; (iii) the actuator moiety complexes with a target linked to a cleavage recognition site; wherein: (i) the cleav polynucleotide after being cleaved from the chimeric recep age recognition site is cleavable by a cleavage moiety in tor polypeptide at the cleavage recognition site; and (iv) the response to receptor binding; and (ii) the actuator moiety is chimeric receptor polypeptide does not comprise SEQ ID operable to complex with a target polynucleotide in response NO: 39. to cleavage of the cleavage recognition site. In some 0035. In some embodiments, the cleavage recognition embodiments, the actuator moiety is operable to translocate site is flanked by the antigen interacting domain and the to a cell nucleus after cleavage of the cleavage recognition actuator moiety. Sequence. 0036. In some embodiments, the antigen interacting 0044. In some embodiments, the actuator moiety is a Cas domain forms a portion of an extracellular region of the protein that forms a complex with a guide RNA. In some chimeric receptor polypeptide, and the GMP forms a portion embodiments, the actuator moiety is an RNA binding pro of an intracellular region of the chimeric receptor polypep tein (RBP) optionally complexed with a guide RNA that is tide. able to form a complex with a Cas protein. In some 0037. In some embodiments, the actuator moiety trans embodiments, the Cas protein substantially lacks DNA locates to a cell nucleus after cleavage of the cleavage cleavage activity. recognition sequence. In some embodiments, the chimeric 0045. In some embodiments, the cleavage recognition receptor polypeptide is a nuclear receptor that translocates to site comprises a polypeptide sequence that is a recognition a cell nucleus in response to antigen binding. sequence of a protease. In some embodiments, the cleavage 0038. In some embodiments, the actuator moiety is a Cas recognition site comprises a first portion of an intein protein that forms a complex with a guide RNA. In some sequence that reacts with a second portion of the intein embodiments, the actuator moiety is an RNA binding pro sequence to release the actuator moiety. In some embodi tein (RBP) optionally complexed with a guide RNA that is ments, the cleavage recognition site comprises a disulfide able to form a complex with a Cas protein. In some bond. embodiments, the Cas protein substantially lacks DNA 0046. In some embodiments, the actuator moiety regu cleavage activity. lates expression of the target polynucleotide by physical 0039. In some embodiments, the cleavage recognition obstruction of the target polynucleotide or recruitment of site comprises a polypeptide sequence that is a recognition additional factors effective to Suppress or enhance expres sequence of a protease. In some embodiments, the cleavage sion of the target polynucleotide. In some embodiments, the recognition site comprises a first portion of an intein actuator moiety comprises an activator effective to increase sequence that reacts with a second portion of the intein expression of the target polynucleotide. In some embodi sequence to release the actuator moiety. In some embodi ments, the actuator moiety is linked to at least one nuclear ments, the cleavage recognition site comprises a disulfide localization signal (NLS). bond. 0047. In some embodiments, the adaptor polypeptide is 0040. In some embodiments, the receptor is a transmem linked to at least one targeting sequence which directs brane receptor. In some embodiments, the receptor is a transport of the adaptor to a specific region of a cell. In some nuclear receptor. embodiments, the targeting sequence directs transport of the 0041. In some embodiments, the actuator moiety regu adaptor to nucleus, cytoplasm, mitochondria, endoplasmic lates expression of the target polynucleotide by physical reticulum (ER), chloroplast, apoplast, peroxisome or plasma obstruction of the target polynucleotide or recruitment of membrane. In some embodiments, the targeting sequence additional factors effective to Suppress or enhance expres comprises a nuclear export signal (NES). In some embodi sion of the target polynucleotide. In some embodiments, the ments, the targeting sequence comprises plasma membrane actuator moiety comprises an activator effective to increase targeting peptide. US 2017/O 1983O8 A1 Jul. 13, 2017

0048. In an aspect, the present disclosure provides a Some embodiments, trasmembrane domain comprises an system for regulating expression of a target polynucleotide amino acid sequence having at least 80%, 81%, 82%, 83%, in a cell. In some embodiments, the system comprises (a) a 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, chimeric receptor polypeptide that is modified upon binding 94%. 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ an antigen, wherein receptor modification comprises a con ID NO:39 or a fragment thereof. In some embodiments, the formational change or chemical modification; (b) a chimeric Cas protein substantially lacks DNA cleavage activity. In adaptor polypeptide that binds the receptor in response to the Some embodiments, the Cas protein is a Cas9 protein. receptor modification; (c) an actuator moiety linked to a 0051. In some embodiments, the gene modulation peptide cleavage domain, wherein upon cleavage of the domain further comprises an activator domain effective to peptide cleavage domain, the actuator moiety is activated to increase expression of a target polynucleotide. In some complex with a target polynucleotide; and (d) a cleavage embodiments, the gene modulation domain further com moiety that cleaves the peptide cleavage domain when in prises a repressor domain effective to decrease expression of proximity to the peptide cleavage domain; wherein: (i) the a target polynucleotide. cleavage moiety forms an intracellular portion of the recep 0052. In some embodiments, the gene modulation tor, and the actuator moiety linked to the peptide cleavage domain further comprises at least one targeting sequence domain forms a portion of the chimeric adaptor polypeptide; which directs transport of the gene modulation domain to a (ii) the cleavage moiety is complexed with a second adaptor specific region of a cell after the gene modulation domain is polypeptide that binds the receptor in response to the recep released from the receptor. In some embodiments, the at tor modification, and the actuator moiety linked to the least one targeting sequences comprises a nuclear localiza peptide cleavage domain forms a portion of the chimeric tion sequence (NLS). adaptor polypeptide; or (iii) the cleavage moiety forms a 0053. In some embodiments, the receptor is linked to at portion of the adaptor polypeptide, and the actuator moiety least one targeting sequence which directs transport of the linked to peptide cleavage domain forms an intracellular receptor to a specific region of a cell. In some embodiments, portion of the receptor. the receptor is linked to a polypeptide folding domain. 0049. In an aspect, the present disclosure provides a 0054. In some embodiments, the peptide cleavage system for regulating expression of a target polynucleotide domain comprises a recognition sequence of a protease. in a cell. In some embodiments, the system comprises (a) a chimeric receptor polypeptide receptor that is modified upon INCORPORATION BY REFERENCE binding an antigen, wherein receptor modification comprises 0055 All publications, patents, and patent applications a conformational change or chemical modification; (b) an chimeric adaptor polypeptide that binds the receptor in mentioned in this specification are herein incorporated by response to the receptor modification; (c) an actuator moiety reference to the same extent as if each individual publica linked to a peptide cleavage domain, wherein upon cleavage tion, patent, or patent application was specifically and indi of the peptide cleavage domain, the actuator moiety is vidually indicated to be incorporated by reference. activated to complex with a target polynucleotide; and (d) a BRIEF DESCRIPTION OF THE DRAWINGS recombinant protease domain that cleaves the peptide cleav age domain when in proximity to the peptide cleavage 0056. The novel features of the invention are set forth domain; wherein: (i) the recombinant protease domain forms with particularity in the appended claims. A better under an intracellular portion of the receptor, and the actuator standing of the features and advantages of the present moiety linked to the peptide cleavage domain forms a invention will be obtained by reference to the following portion of the chimeric adaptor polypeptide; (ii) the recom detailed description that sets forth illustrative embodiments, binant protease domain is complexed with a second adaptor in which the principles of the invention are utilized, and the polypeptide that binds the receptor in response to the recep accompanying drawings of which: tor modification, and the actuator moiety linked to the 0057 FIG. 1 shows an exemplary chimeric receptor peptide cleavage domain forms a portion of the chimeric polypeptide comprising an antigen interacting domain and a adaptor polypeptide; or (iii) the recombinant protease gene modulating polypeptide (GMP). domain forms a portion of the chimeric adaptor polypeptide, 0.058 FIG. 2 shows an exemplary chimeric transmem and the actuator moiety linked to the peptide cleavage brane receptor polypeptide. domain forms an intracellular portion of the receptor. 0059 FIG. 3A shows an exemplary chimeric receptor 0050. In an aspect, the present disclosure provides a polypeptide including an actuator moiety comprising an chimeric receptor polypeptide. The chimeric receptor poly RNA-binding protein optionally complexed to a guide peptide comprises: an extracellular antigen interacting nucleic acid (e.g., sgRNA). FIG. 3B shows an exemplary domain which binds an antigen; a transmembrane domain; system comprising a chimeric receptor polypeptide and a and an intracellular gene modulation domain comprising a chimeric adaptor polypeptide comprising a cleavage moiety. Cas protein, wherein a peptide cleavage domain is located at 0060 FIGS. 4A-D illustrate schematically the release of the amino terminus of the gene modulation domain; wherein an actuator moiety from a GMP in a system comprising a upon binding of the extracellular antigen interacting domain receptor which undergoes phosphorylation; FIGS. 4E-H to the antigen, the gene modulation domain is released from illustrate schematically the release of an actuator moiety the chimeric receptor polypeptide by cleavage of the peptide from a GMP in a system comprising a receptor which cleavage domain. In some embodiments, the chimeric recep undergoes a conformational change. tor polypeptide undergoes a receptor modification upon 0061 FIG. 5 shows an exemplary chimeric receptor binding to the antigen. In some embodiments, the transmem polypeptide comprising at least one targeting sequence. brane domain comprises a portion of a Notch receptor 0062 FIG. 6A shows an exemplary chimeric adaptor protein, or any derivative, variant, or fragment thereof. In polypeptide comprising a receptor binding moiety and a US 2017/O 1983O8 A1 Jul. 13, 2017 gene modulating polypeptide (GMP). FIG. 6B shows an gene modulating effector domain. The other receptor of the exemplary chimeric adaptor polypeptide including an actua dimer includes an ECD, TM, ICD, and a protease. FIG. 19E tor moiety comprising an RNA-binding protein optionally shows another example of dimerizing receptors that can complexed to a guide nucleic acid (e.g., sgRNA). modulate gene expression or edit genes. One of the dimeriZ 0063 FIG. 7 shows an exemplary system comprising a ing receptors can include an ECD, TM, ICD, peptide chimeric receptor polypeptide comprising a cleavage moiety cleavage sequence, and a gene modulating effector domain. and a chimeric adaptor polypeptide comprising a GMP. The other receptor of the dimer can include an ECD, TM and 0064 FIGS. 8A-D illustrate schematically the release of ICD, and not a protease. The protease that cleaves this an actuator moiety from a GMP in a system comprising a dimerizing receptor can be fused to an adaptor protein that receptor which undergoes phosphorylation; FIGS. 8E-H associates to the activated dimerizing receptor. FIG. 19F illustrate schematically the release of an actuator moiety shows an example of an oligomerizing receptor that includes from a GMP in a system comprising a receptor which engineered chimeric antigen receptors fused to gene modu undergoes a conformational change. lation domains. 0065 FIG. 9 shows an exemplary system comprising a (0076 FIGS. 20A and 20B provides different chimeric chimeric receptor polypeptide, a chimeric adaptor polypep antigen receptors and illustrates the binding of a dCas9 tide comprising a GMP, and a second adaptor polypeptide activator domain guided to a target gene by an SgRNA. FIG. comprising a cleavage moiety. 20A shows recombinant chimeric antigen receptor polypep 0066 FIGS. 10A-D illustrate schematically the release of tides and in some cases, their associated adaptor-protease an actuator moiety from a GMP in a system comprising at polypeptides Such as Notch and presenillin-proteases, least two adaptor polypeptides and a receptor which under GPCRs and B2-arrestin-proteases, integrins and paxillin goes phosphorylation: FIGS. 10E-H illustrate schematically proteases, cadherins and B-catenin-proteases, death recep the release of an actuator moiety from a GMP in a system tors and FADD-proteases, and chimeric antigen receptors. comprising at least two adaptor polypeptides and a receptor (0077 FIG. 21A show uses of chimeric antigen GPCRs which undergoes a conformational change. coupled to dCas9-activators. FIG. 21B illustrates an alter 0067 FIG. 11 shows an exemplary chimeric adaptor native configuration in which the protease moiety is coupled polypeptide comprising at least one targeting sequence. to the GPCR and the dCas9-activator domain is coupled to 0068 FIGS. 12A-C illustrate schematically a system an adaptor protein recruited to an activated GPCR. comprising an exemplary intracellular receptor. (0078 FIGS. 22A and 22B show integrin-dCas'9 gene 0069 FIGS. 13 A-D illustrate schematically a system in modulating polypeptides and their response to integrin which the cleavage recognition site comprises an intein ligands such as fibronectin. FIG. 22A depicts a schematic sequence: FIGS. 13E-Hillustrate an alternative arrangement diagram of a chimeric antigen integrin-dCas9 activator. FIG. of a system in which the cleavage recognition site comprises 22B shows an integrin-dCas9 complex that is responsive to an intein sequence. fibronectin. Upon binding to an sgRNA specific to the 0070 FIGS. 14A-D illustrate schematically a system in reporter gene, the recombinant complex activated transcrip which the cleavage recognition site comprises a disulfide tion of the reporter (H2B-GFP). FIG. 22C illustrates the bond; FIGS. 14E-H illustrate an alternative arrangement of activity of integrin-dCas9 complex in adherent cells com a system in which the cleavage recognition site comprises a pared to suspension cells. FIGS. 22D and 22E illustrates the disulfide bond. binding specificity of paxillin-TEV for the beta subunit of (0071 FIG. 15 shows an illustration adapted from FIG. 2 integrin relative to the alpha Subunit of Makarova, K. S. et al., “An updated evolutionary classi (0079 FIGS. 23A and 23B provide an exemplary embodi fication of CRISPR-Cas systems.” Nat Rev Microbiol ment of chimeric GPCR-gene modulating domain polypep (2015) 13:722-736 providing architectures of the genomic tide. FIG. 23A shows a scheme of target gene regulation by loci for subtypes of CRISPR-Cas systems. GPCR based chimeric antigen receptor-dCas9 activators. 0072 FIGS. 16A-D illustrate schematically the release of FIG. 23B shows that the CXCR4-dCas9 polypeptide was an actuator moiety from a GMP in a system comprising at responsive to CXCL 12 and activated the luminescent least two adaptor polypeptides. reporter. FIGS. 23C and 23D illustrate chimeric GPCR 0073 FIG. 17 shows a schematic diagram of engineered receptors comprising LPAR1, CXCR4, and hM3D and cor chimeric antigen receptors of the present invention for gene responding activation of a fluorescent reporter (GFP) in the modulation Such as genome editing and gene regulation. presence of ligand, B-arrestin-protease, and sgRNA. FIGS. 0074 FIG. 18 shows variants of linkers located between 23E and 23F compare levels of transcriptional regulation of the transmembrane domain and the gene modulation domain a reporter gene resulting from dCas9-VPR targeted by of the engineered chimeric antigen receptors of the present sgRNA after release from a chimeric receptor and a TetR disclosure. VPR which binds directly to the promoter of the reporter 0075 FIGS. 19A-19F show engineered chimeric antigen gene. receptors with gene modulation domains and in some cases, 0080 FIGS. 24A-24G show exemplary embodiments of their associated adaptor-proteases. FIG. 19A depicts such modular chimeric artificial Notch receptors of the present recombinant receptors that bind to cell Surface antigens. invention. FIG. 24A shows a wild-type Notch bound to its FIG. 19B depicts recombinant receptors for gene modula ligand Delta. After the receptor is activated by binding tion that can bind soluble antigens. FIG. 19C illustrates gene Delta, the ICD is cleaved by a protease and translocates to modulating, engineered receptors that can bind to extracel the nucleus to regulate target genes. FIG. 24B shows a lular matrix (ECM) signals. FIG. 19D illustrates dimerizing chimeric artificial Notch receptor where the Notch ICD has receptors. One of the receptors includes an extracellular been replaced with a dCas9 fusion protein. The dCas9 fusion domain (ECD), a transmembrane domain (TM), an intrac protein can include an effector domain such as an activator ellular domain (ICD), a peptide-cleavage sequence, and a domain, e.g., VP64 domain or a repressor domain, e.g., US 2017/O 1983O8 A1 Jul. 13, 2017

KRAB domain. FIG. 24C shows another chimeric artificial integrin-based engineered chimeric antigen receptor-dCas9 Notch receptor containing a dCas9 fusion protein where the complex induced reporter expression in response to signals Notch ECD has been replaced with a CD47-binding schv. from the ECM. FIG. 24D shows an exemplary modular chimeric artificial I0086 FIGS. 30A and 30B show exemplary embodiments Notch receptor and an adapter-protease fusion protein (pres of the chimeric antigen receptor-effector polypeptides inillin-TEV protease) expressed on the surface of a cell such described herein that are based on a “Split AND gate' or as an immune cell. The modular chimeric artificial Notch “Cascade AND gate' logic. FIG. 30A shows a schematic receptor can contain a dCas9 fusion polypeptide, a linker, diagram of a split dOas9 effector tethered to separate engi and an effector domain. Upon Delta-Notch binding, the neered receptors. FIG. 30B provides a schematic diagram of presinillin can associate with the chimeric artificial Notch a chimeric receptor-tTa polypeptide that, upon binding to its receptor. Then, the TEV protease can cleave the peptide ligand, induces expression of a TetO-driven chimeric recep cleavage domain of the chimeric artificial Notch receptor. tor-dCas9 polypeptide. Activation of a fluorescent reporter in cells expressing the I0087 FIGS. 31A-31I illustrate embodiments of a recep Notch-dCas'9-activator is shown in FIG. 24E for HEK293 tor-based strategy to mobilize Cas9 in response to extracel cells, in FIG. 24F for Jurkat cells, and in FIG.24G in THP-1 lular signals. FIG. 31A illustrates activation of Notch1 macrophages. receptors involving cleavage and nuclear translocation of the 0081 FIGS. 25A-25C show theoretical models for Notch intracellular domain, which can be replaced by or reshaping the endogenous response of the Notch receptor engineered to promote expression of Cas9 derivatives. The using the chimeric antigen Notch receptors described herein. fusion of effector domains to Cas9 and a user-defined FIG. 25A shows the repression of the endogenous phago single-guide RNA (sgRNA) sequence allow for targeted cytic response of a receiving cell expressing endogenous gene regulation. FIG. 31B shows Schematic designs of Notch upon binding Delta expressed on a signaling cell. mCherry-tagged chimeric receptor constructs that were ini FIG. 25B shows that the engineered caN receptor can be tially tested for cellular localization and Delta-dependent created to rewire the endogenous phagocytic response to an reporter activation. The human codon-optimized nuclease external Delta signal. FIG. 25C shows that the engineered dead Cas9 (dCas9) and tripartite activator domains (VP64, caN receptor can be produced to shift the repression of the p65, and Rita; VPR) are fused immediately after the Notch1 cells endogenous phagocytic response to activation upon extracellular domain (hNECD) and transmembrane domain. Delta binding. Construct NC5 comprises maturation signals derived from a known ER export signal. FIG. 31C shows a schematic of a 0082 FIGS. 26A-26D show that the Notch-dCas9 acti Chinese hamster ovary (CHO) cell line integrated with an vator, upon Delta binding, can activate target genes Such as Upstream Activating Sequence (UAS) or CSL-binding (not those that control cell apoptosis or the cell cycle. Cells shown) promoters that drive a Histone 2B (H2B)-citrine expressing the Notch-dCas9 polypeptide activated the target reporter gene and a stably integrated promoter-targeting genes when in the presence of Delta (FIG. 26B and 26D). sgRNA (e.g., SgUAS or sasgCSL, respectively) used to Notch chimeric antigen receptor did not activate transcrip validate gene-activation efficiency of chimeric receptors tion of the target genes in the absence of Delta (FIGS. 26A when cultured with or without surface-immobilized Delta. and 26C). Activation of NC5 receptors by immobilized Delta ligands 0083 FIGS. 27A and 27B show that the Notch-dCas9 leads to cleavage and nuclear translocation of dCas9-VPR. activator is guided by a sgUAS (SEQ ID NO:1; gtacticcgac dCas'9-VPR complexed with a sequence-specific sgRNA ctictagtgt) to a UAS promoter and activates transcription of (e.g., SgUAS or sasgOSL) allows for binding of the complex a reporter gene (H2B-citrine). FIG. 27A provides a sche to the promoter and activation of H2B-citrine gene. FIG. matic diagram of the process. FIG. 27B shows that the 31D shows example microscopy images of CHO cells Notch-dCas9 activator is responsive to Delta. transfected with the NC5 chimera resulting in H2B expres sion when exposed to immobilized Delta for 4 days. Scale 0084 FIGS. 28A and 28B show that the CXCR4-dCas9 bar, 20 um. FIG. 31E shows relative H2B levels (normalized VPR polypeptide is responsive to CXCL12 ligand and expression) in CHO UAS-H2B clones stably selected for activates transcription of a reporter gene (luciferase). FIG. NC5 (S. pyogenes dCas'9, and sgUAS) or in CHO CSL-H2B 28A provides a schematic diagram of ligand binding of the clones with wild-type human Notch1 or NC5 (S. aureus CXCR4-dCas9-VPR polypeptide that is complexed with a dCas'9, and sasgCSL) and cultured on bare or immobilized sgRNA (sgTET, SEQ ID NO:2; gtacgttctictatcactgata). FIG. Delta surfaces for 4 days (n=3). MeaniSEM. FIG. 31F 28A also shows a B-arrestin-protease fusion protein that can shows percentage of cells that activate H2B-citrine in CHO associated with the engineered chimeric antigen receptor. UAS-H2B clones stably selected for NC5 (S. pyogenes The diagram also shows (1) translocation of free dCas9 dCas9, and sgUAS) or in CHO 12xCSL-H2B clones with VPR into the nucleus, (2) binding of the sgTET-dCas9-VPR wild-type human Notch1 or NC5 (S. aureus dCas9, and complex to a TetO promoter that regulates transcription of sasgCSL) and cultured on bare or immobilized-Delta sur the luciferase gene, and (3) transcription of the reporter. FIG. faces for 4 days (n=3). MeaniSEM, **p<0.01, compared to 28B shows that transcription of luciferase gene is regulated (-Delta) controls. FIG. 31G illustrates activation of NC5 by CXCL2 binding to the CXCR4-dCas'9-VPR polypeptide. receptors by immobilized Delta ligand resulting in cleavage I0085 FIGS. 29A and 29B show that the integrin-dCas9 and nuclear translocation of dCas'9-VPR. FIG. 31H shows VPR polypeptide is responsive to an extracellular matrix EGFP reporter intensity historgrams from HEK293T ligand and activates transcription of a reporter gene (H2B reporter cells stably expressing a tet-inducible EGFP gene citrine). FIG. 29.A shows a schematic diagram of transcrip and a targeting sgRNA (sgTET) in the presence of dCas9 tional activation of the reporter upon ligand binding to the VPR, NC5+Delta--DAPT, NC5+Delta, NC5-Delta, and no integrin-dCas9-VPR polypeptide. FIG. 29B shows that the construct. FIG. 31I shows contour plots (where the same US 2017/O 1983O8 A1 Jul. 13, 2017

number of cells fall between each pair of contour lines) of schematic for two sgRNAs (short bars to left and right of EGFP activation of HEK293T reporter cells transfected with “sgCXCR4') that were stably expressed in HEK293T cells NC5 receptor and cultured on various concentrations of and were designed to target the 5' untranslated region (UTR) immobilized Delta for 3 days. and intron 1 of CXCR4. Scale bar, 1000 bp. FIG. 36D, top 0088 FIG. 32A illustrates a schematic of a synthetic panel, shows example results from a T7E1 endonuclease to Cas9-receptor System in accordance with an embodiment assay the extent of Delta-induced hNECD-Cas'9-mediated described herein. Modulation of target endogenous genes is modification of CXCR4 gene in HEK293T cells, as detected responsive to extracellular signals, such as Delta. A variety by amount of products cleaved by T7E1 in SDS-PAGE gels. of downstream cellular behaviors can be triggered from The bottom panel shows example results for frequency of native extracellular inputs, depending on design of the CXCR4 indel mutations. FIG. 36E shows example results system. FIG. 32B left shows CDKN1B activation via for the quantification of flow cytometry-based immunofluo hNECD-dCas'9-VPR (construct NC5) induces a Delta-de rescence staining of CXCR4 protein expression in pendent cell-cycle arrest at G0/G1 phase. Right shows a HEK293T cells. schematic representation of Delta-dependent cleavage of (0093 FIG. 37A shows alignment of EGF-11 and -12 dCas9-VPR from NC5 leading to CDKN1B-high and repeats of human, Xenopus, Zebrafish, and Drosophila G0/G1-arrested cells (with 2n DNA), in accordance with an homologs (SEQ ID NO: 57-60). Identical residues are embodiment. FIG.32C shows example flow-cytometry plots indicated in gray boxes. Asterisk (*) marks conserved cys and percentage quantification (shaded regions) of teine and Ca"-binding consensus residues. FIG. 37B shows CDKN1B-high and G0/G1-arrested cells under different variable activation levels of various EGF deletion variants culture conditions: HEK293 cells with sgRNA only, (as evaluated by reporter assay). sgRNA--Delta, sgRNA-i-NC5, or sgRNA-i-NC5+Delta. NC5 0094 FIG. 38 shows the simultaneous activation of was transiently transfected in sgCDKN1B-integrated cells, CXCR4 and CD95 genes targeted by gene-specific sgRNAs which were then cultured on Delta-coated or bare surfaces (2 per gene). Relative activation levels of CXCR4 and CD95 for 4 days. n=3, Mean-SEM. G0/G1 arrested cells corre under indicated conditions in 4-day cultures are shown (a.u., spond to the top left shaded corner of the plots, and the top arbitrary units). Data is displayed as mean fluorescence bar of each pair in the bar graph. G2 arrested cells corre intensity-SEM (n=3 independent experiments). i.iiip-0.01, spond to the top right shaded corner of the plots, and bottom 0.001, *, **p-0.01, 0.001, compared to CXCR4 and CD95 bar of each pair in the bar graph. levels, respectively in negative control. dCas9-VPR+sgNT 0089 FIG. 33 left panel shows an example confocal (non-targeting). fluorescence micrograph of a CHO cell transfected with 0095 FIGS. 39A-39F show the conversion of Delta construct NC1 (red) and sgUAS (blue). Premature activation signals to cell cycle phase-specific arrest. FIG. 39A illus of H2B (green, nucleus) is observed. Right panel shows a trates schematically the use of minimal NC5 receptor variant schematic representation of balance between the strengths of to elicit Delta-dependent arrest of the cell cycle. FIG. 39B nuclear localization signals and membrane maturation sig depicts cellular arrest at the G0/G1 phase resulting from nals in Delta-dependent cleavage of Notch-Cas9 chimeras. CDKN1B overexpression. FIG. 39C and 39D shows that in 0090 FIG. 34 shows fluorescence microscopy images of cells with dCas'9-VPR and sgCDKN1B, CDKN1B upregu CHO cells transfected with constructs NC1-NC4 (top, see lation was concomitant with G0/G1 enrichment, and in cells FIG. 31B) and having H2B expression (bottom) in the with dCas'9-VPR and non-targeting gskNA, a minimal absence of Delta. Scale bar, 20 Lum. increase in CDKN1B was observed. FIGS. 39E and 39F 0091 FIG. 35A shows a naturally occurring NLS show abrogation of Delta-induced CDKN1B upregulation sequence in S. py. Cas9 found at amino acid residues and G0/G1 arrest in cells with DAPT. 647-670. The illustration shows this intrinsic NLS (iNLS) can form a surface-accessible helix-linker-helix structure DETAILED DESCRIPTION (PDB ID: 4UN3) (SEQ ID NO:33, 34). FIG. 35B shows 0096. The practice of some methods disclosed herein example results for HEK293 cells stably integrated with employ, unless otherwise indicated, conventional techniques pTET-EGFP reporter and targeting sgRNA (sgTET) that of immunology, biochemistry, chemistry, molecular biology, were transfected with dCas9-VPR (top) or mutated-iNLS microbiology, cell biology, genomics and recombinant dCas'9-VPR (center). Representative histograms of EGFP DNA, which are within the skill of the art. See for example activation in the presence or absence (bottom) of constructs Sambrook and Green, Molecular Cloning: A Laboratory and quantification of EGFP activation (normalized to Manual, 4th Edition (2012); the series Current Protocols in untransfected cells) is shown (SEQ ID NO: 31, 32). FIGS. Molecular Biology (F. M. Ausubel, et al. eds.); the series 35C shows by microscopy impaired nuclear localization and Methods In Enzymology (Academic Press, Inc.), PCR 2: A EGFP reporter activation in HEK293T cells when the iNLS Practical Approach (M. J. MacPherson, B. D. Hames and G. motif is disrupted (SEQ ID NO:31, 32, 9). FIGS. 35C and R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) 35D show visually by microscopy and quantitatively that Antibodies, A Laboratory Manual, and Culture of Animal adding a synthetic NLS to the N-terminus of iNLS-mutated Cells: A Manual of Basic Technique and Specialized Appli dCas9-VPR partially restores EGFP activation. cations, 6th Edition (R. I. Freshney, ed. (2010)). 0092 FIGS. 36A-36E show Delta-dependent gene edit 0097. As used in the specification and claims, the singu ing. FIG. 36A shows schematically Delta-dependent DNA lar forms “a”, “an and “the' include plural references cutting with an NC5 variant: hNECD fused to wild-type S. unless the context clearly dictates otherwise. For example, pyogenes nuclease-active Cas9 (hNECD-Cas'9). FIG. 36B the term “a chimeric transmembrane receptor includes a shows the efficacy of gene editing/cutting with hNECD plurality of chimeric transmembrane receptors. Cas9 in CHO cells with stably integrated EGFP and a (0098. The term “about” or “approximately” means within targeting sgRNA (sgEGFP). FIG. 36C shows an example an acceptable error range for the particular value as deter US 2017/O 1983O8 A1 Jul. 13, 2017 mined by one of ordinary skill in the art, which will depend amine (R6G), N.N.N',N'-tetramethyl-6-carboxyrhodamine in part on how the value is measured or determined, i.e., the (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethyl limitations of the measurement system. For example, aminophenylazo) benzoic acid (DABCYL), Cascade Blue, “about can mean within 1 or more than 1 standard devia Oregon Green, Texas Red, Cyanine and 5-(2-aminoethyl) tion, per the practice in the art. Alternatively, “about can aminonaphthalene-1-sulfonic acid (EDANS). Specific mean a range of up to 20%, up to 10%, up to 5%, or up to examples of fluorescently labeled nucleotides can include 1% of a given value. Alternatively, particularly with respect R6GdUTP (TAMRAdUTP. R110dCTP R6G dCTP, to biological systems or processes, the term can mean within (TAMRA dCTP, (JOE) ddATP R6G ddATP, FAM an order of magnitude, preferably within 5-fold, and more ddCTP, R110ddCTP (TAMRAddGTP. ROXddTTP, preferably within 2-fold, of a value. Where particular values dR6GddATP, dR110ddCTP, dTAMRAddGTP, and are described in the application and claims, unless otherwise dROXddTTP available from Perkin Elmer, Foster City, stated, the term “about” meaning within an acceptable error Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3 range for the particular value should be assumed. dCTP. FluoroLink Cy5-dCTP. FluoroLink Fluor X-dCTP, 0099. As used herein, a “cell' can generally refer to a FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP avail biological cell. A cell can be the basic structural, functional able from Amersham, Arlington Heights, Ill.; Fluorescein and/or biological unit of a living organism. A cell can 15-daTP. Fluorescein-12-dUTP. Tetramethyl-rodamine-6- originate from any organism having one or more cells. Some dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein non-limiting examples include: a prokaryotic cell, eukary 12-UTP, and Fluorescein-15-2'-dATP available from otic cell, a bacterial cell, an archaeal cell, a cell of a Boehringer Mannheim, Indianapolis, Ind., and Chromosome single-cell eukaryotic organism, a protozoa cell, a cell from Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4- a plant (e.g. cells from plant crops, fruits, vegetables, grains, UTP BODIPY-TMR-14-UTP BODIPY-TMR-14-dUTP, Soybean, corn, maize, wheat, seeds, tomatoes, rice, cassava, BODIPY-TR-14-UTP BODIPY-TR-14-dUTP. Cascade Sugarcane, pumpkin, hay, potatoes, cotton, cannabis, Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, tobacco, flowering plants, conifers, gymnosperms, ferns, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhod clubmosses, hornworts, liverworts, mosses), an algal cell, amine Green-5-UTP, Rhodamine Green-5-dUTP, tetrameth (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, ylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP sum patens C. Agardh, and the like), seaweeds (e.g. kelp), a available from Molecular Probes, Eugene, Oreg. Nucleo fungal cell (e.g., a yeast cell, a cell from a mushroom), an tides can also be labeled or marked by chemical modifica animal cell, a cell from an invertebrate animal (e.g. fruit fly, tion. A chemically-modified single nucleotide can be biotin cnidarian, echinoderm, nematode, etc.), a cell from a verte dNTP. Some non-limiting examples of biotinylated dNTPs brate animal (e.g., fish, amphibian, reptile, bird, mammal), a can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14 cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a dATP), biotin-dCTP (e.g., biotin-11-dCTP biotin-14-dCTP), rodent, a rat, a mouse, a non-human primate, a human, etc.), and biotin-dUTP (e.g. biotin-11-dUTP biotin-16-dUTP bio and etcetera. Sometimes a cell is not originating from a tin-20-dUTP). natural organism (e.g. a cell can be a synthetically made, 0101 The terms “polynucleotide,” “oligonucleotide.” Sometimes termed an artificial cell). and “nucleic acid are used interchangeably to refer to a 0100. The term “nucleotide,” as used herein, generally polymeric form of nucleotides of any length, either deoxy refers to a base-Sugar-phosphate combination. A nucleotide ribonucleotides or ribonucleotides, or analogs thereof, either can comprise a synthetic nucleotide. A nucleotide can com in single-, double-, or multi-stranded form. A polynucleotide prise a synthetic nucleotide analog. Nucleotides can be can be exogenous or endogenous to a cell. A polynucleotide monomeric units of a nucleic acid sequence (e.g. deoxyri can exist in a cell-free environment. A polynucleotide can be bonucleic acid (DNA) and ribonucleic acid (RNA)). The a gene or fragment thereof. A polynucleotide can be DNA. term nucleotide can include ribonucleoside triphosphates A polynucleotide can be RNA. A polynucleotide can have adenosine triphosphate (ATP), uridine triphosphate (UTP), any three dimensional structure, and can perform any func cytosine triphosphate (CTP), guanosine triphosphate (GTP) tion, known or unknown. A polynucleotide can comprise one and deoxyribonucleoside triphosphates such as dATP, dCTP, or more analogs (e.g. altered backbone, Sugar, or nucle dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such obase). If present, modifications to the nucleotide structure derivatives can include, for example, C.SdATP, 7-deaza can be imparted before or after assembly of the polymer. dGTP and 7-deaza-dATP, and nucleotide derivatives that Some non-limiting examples of analogs include: 5-bromou confer nuclease resistance on the nucleic acid molecule racil, peptide nucleic acid, Xeno nucleic acid, morpholinos, containing them. The term nucleotide as used herein can locked nucleic acids, glycol nucleic acids, threose nucleic refer to dideoxyribonucleoside triphosphates (ddNTPs) and acids, dideoxynucleotides, cordycepin, 7-deaza-GTP floro their derivatives. Illustrative examples of dideoxyribo phores (e.g. rhodamine or flurescein linked to the Sugar), nucleoside triphosphates can include, but are not limited to, thiol containing nucleotides, biotin linked nucleotides, fluo ddATP, ddCTP, ddGTP, dd ITP, and ddTTP. A nucleotide rescent base analogs, CpG islands, methyl-7-guanosine, may be unlabeled or detectably labeled by well-known methylated nucleotides, inosine, thiouridine, pseudourdine, techniques. Labeling can also be carried out with quantum dihydrouridine, queuosine, and Wyosine. Non-limiting dots. Detectable labels can include, for example, radioactive examples of polynucleotides include coding or non-coding isotopes, fluorescent labels, chemiluminescent labels, bio regions of a gene or gene fragment, loci (locus) defined from luminescent labels and enzyme labels. Fluorescent labels of linkage analysis, exons, introns, messenger RNA (mRNA), nucleotides may include but are not limited fluorescein, transfer RNA (tRNA), ribosomal RNA (rRNA), short inter 5-carboxyfluorescein (FAM), 27'-dimethoxy-4,5-dichloro fering RNA (siRNA), short-hairpin RNA (shRNA), micro 6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhod RNA (miRNA), ribozymes, cDNA, recombinant polynucle US 2017/O 1983O8 A1 Jul. 13, 2017

otides, branched polynucleotides, plasmids, vectors, isolated portions thereof. See, e.g., Sambrook et al., 1989, Molecular DNA of any sequence, isolated RNA of any sequence, Cloning: A Laboratory Manual, 18.1-18.88. cell-free polynucleotides including cell-free DNA (cfloNA) 0105. The term “expression” refers to one or more pro and cell-free RNA (cfRNA), nucleic acid probes, and prim cesses by which a polynucleotide is transcribed from a DNA ers. The sequence of nucleotides can be interrupted by template (such as into an mRNA or other RNA transcript) non-nucleotide components. and/or the process by which a transcribed mRNA is subse 0102 The terms “target polynucleotide' and “target quently translated into peptides, polypeptides, or proteins. nucleic acid,” as used herein, refer to a nucleic acid or Transcripts and encoded polypeptides can be collectively polynucleotide which is targeted by an actuator moiety of referred to as “gene product.” If the polynucleotide is the present disclosure. A target nucleic acid can be DNA. A derived from genomic DNA, expression can include splicing target nucleic acid can be RNA. A target nucleic acid can of the mRNA in a eukaryotic cell. “Up-regulated, with refer to a chromosomal sequence or an extrachromosomal reference to expression, generally refers to an increased sequence, (e.g., an episomal sequence, a minicircle expression level of a polynucleotide (e.g., RNA Such as sequence, a mitochondrial sequence, a chloroplast sequence, mRNA) and/or polypeptide sequence relative to its expres etc.). A target nucleic acid can be a nucleic acid sequence sion level in a wild-type state while “down-regulated that may not be related to any other sequence in a nucleic generally refers to a decreased expression level of a poly acid sample by a single nucleotide Substitution. A target nucleotide (e.g., RNA such as mRNA) and/or polypeptide nucleic acid can be a nucleic acid sequence that may not be sequence relative to its expression in a wild-type state. related to any other sequence in a nucleic acid sample by a Expression of a transfected gene can occur transiently or 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some stably in a cell. During “transient expression the transfected embodiments, the substitution may not occur within 5, 10. gene is not transferred to the daughter cell during cell 15, 20, 25, 30, or 35 nucleotides of the 5' end of a target division. Since its expression is restricted to the transfected nucleic acid. In some embodiments, the Substitution may not cell, expression of the gene is lost over time. In contrast, occur within 5, 10, 15, 20, 25, 30, 35 nucleotides of the 3' stable expression of a transfected gene can occur when the end of a target nucleic acid. In general, the term “target gene is co-transfected with another gene that confers a sequence” refers to a nucleic acid sequence on a single selection advantage to the transfected cell. Such a selection Strand of a target nucleic acid. The target sequence can be a advantage may be a resistance towards a certain toxin that is portion of a gene, a regulatory sequence, genomic DNA, cell presented to the cell. free nucleic acid including cfLNA and/or cfRNA, cDNA, a 0106 The term "expression cassette,” “expression con fusion gene, and RNA including mRNA, miRNA, rRNA, struct, or “expression vector” refers to a nucleic acid that and others. includes a nucleotide sequence Such as a coding sequence 0103) The term “gene.” as used herein, refers to a nucleic and a template sequence, and sequences necessary for acid (e.g., DNA such as genomic DNA and cDNA) and its expression of the coding sequence. The expression cassette corresponding nucleotide sequence that is involved in can be viral or non-viral. For instance, an expression cassette encoding an RNA transcript. The term as used herein with includes a nucleic acid construct, which when introduced reference to genomic DNA includes intervening, non-coding into a host cell, results in transcription and/or translation of regions as well as regulatory regions and can include 5' and a RNA or polypeptide, respectively. Antisense constructs or 3' ends. In some uses, the term encompasses the transcribed sense constructs that are not or cannot be translated are sequences, including 5' and 3' untranslated regions (5'-UTR expressly included by this definition. One of skill will and 3'-UTR), exons and introns. In some genes, the tran recognize that the inserted polynucleotide sequence need not scribed region will contain "open reading frames' that be identical, but may be only substantially similar to a encode polypeptides. In some uses of the term, a "gene’ sequence of the gene from which it was derived. comprises only the coding sequences (e.g., an “open reading 0107. A "plasmid, as used herein, generally refers to a frame' or "coding region') necessary for encoding a poly non-viral expression vector, e.g., a nucleic acid molecule peptide. In some cases, genes do not encode a polypeptide, that encodes for genes and/or regulatory elements necessary for example, ribosomal RNA genes (rRNA) and transfer for the expression of genes. A “viral vector, as used herein, RNA (tRNA) genes. In some cases, the term “gene' includes generally refers to a viral-derived nucleic acid that is capable not only the transcribed sequences, but in addition, also of transporting another nucleic acid into a cell. A viral vector includes non-transcribed regions including upstream and is capable of directing expression of a protein or proteins downstream regulatory regions, enhancers and promoters. A encoded by one or more genes carried by the vector when it gene can refer to an "endogenous gene' or a native gene in is present in the appropriate environment. Examples for viral its natural location in the genome of an organism. A gene can vectors include, but are not limited to retroviral, adenoviral, refer to an “exogenous gene' or a non-native gene. A lentiviral and adeno-associated viral vectors. non-native gene can refer to a gene not normally found in the 0108. The term “promoter,” as used herein, refers to a host organism but which is introduced into the host organism polynucleotide sequence capable of driving transcription of by gene transfer. A non-native gene can also refer to a gene a coding sequence in a cell. Thus, promoters used in the not in its natural location in the genome of an organism. A polynucleotide constructs of the disclosure include cis non-native gene can also refer to a naturally occurring acting transcriptional control elements and regulatory nucleic acid or polypeptide sequence that comprises muta sequences that are involved in regulating or modulating the tions, insertions and/or deletions (e.g., non-native sequence). timing and/or rate of transcription of a gene. For example, a 0104. The terms “transfection' or “transfected refer to promoter can be a cis-acting transcriptional control element, introduction of a nucleic acid into a cell by non-viral or including an enhancer, a promoter, a transcription termina viral-based methods. The nucleic acid molecules may be tor, an origin of replication, a chromosomal integration gene sequences encoding complete proteins or functional sequence, 5' and 3' untranslated regions, or an intronic US 2017/O 1983O8 A1 Jul. 13, 2017 sequence, which are involved in transcriptional regulation. is it intended to imply or distinguish whether the peptide is These cis-acting sequences typically interact with proteins produced using recombinant techniques, chemical or enzy or other biomolecules to carry out (turn on/off, regulate, matic synthesis, or is naturally occurring. The terms apply to modulate, etc.) gene transcription. A "constitutive promoter” naturally occurring amino acid polymers as well as amino is one that is capable of initiating transcription in nearly all acid polymers comprising at least one modified amino acid. tissue types, whereas a “tissue-specific promoter initiates In some cases, the polymer can be interrupted by non-amino transcription only in one or a few particular tissue types. An acids. The terms include amino acid chains of any length, “inducible promoter is one that initiates transcription only including full length proteins, and proteins with or without under particular environmental conditions, developmental secondary and/or tertiary structure (e.g., domains). The conditions, or drug or chemical conditions. terms also encompass an amino acid polymer that has been 0109. The terms “complement,” “complements.” modified, for example, by disulfide bond formation, glyco “complementary,” and "complementarity,” as used herein, Sylation, lipidation, acetylation, phosphorylation, oxidation, generally refer to a sequence that is fully complementary to and any other manipulation Such as conjugation with a and hybridizable to the given sequence. In some cases, a labeling component. The terms 'amino acid and "amino sequence hybridized with a given nucleic acid is referred to acids, as used herein, generally refer to natural and non as the “complement' or “reverse-complement of the given natural amino acids, including, but not limited to, modified molecule if its sequence of bases over a given region is amino acids and amino acid analogues. Modified amino capable of complementarily binding those of its binding acids can include natural amino acids and non-natural amino partner, such that, for example, A-T, A-U, G-C, and G-U acids, which have been chemically modified to include a base pairs are formed. In general, a first sequence that is group or a chemical moiety not naturally present on the hybridizable to a second sequence is specifically or selec amino acid. Amino acid analogues can refer to amino acid tively hybridizable to the second sequence, such that hybrid derivatives. The term "amino acid' includes both D-amino ization to the second sequence or set of second sequences is acids and L-amino acids. preferred (e.g. thermodynamically more stable under a given (O112 The terms “derivative,” “variant,” and “fragment,” set of conditions, such as stringent conditions commonly when used herein with reference to a polypeptide, refers to used in the art) to hybridization with non-target sequences a polypeptide related to a wild type polypeptide, for example during a hybridization reaction. Typically, hybridizable either by amino acid sequence, structure (e.g., secondary sequences share a degree of sequence complementarity over and/or tertiary), activity (e.g., enzymatic activity) and/or all or a portion of their respective lengths, such as between function. Derivatives, variants and fragments of a polypep 25%-100% complementarity, including at least 25%, 30%, tide can comprise one or more amino acid variations (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, mutations, insertions, and deletions), truncations, modifica 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, tions, or combinations thereof compared to a wild type 99%, and 100% sequence complementarity. Sequence iden polypeptide. tity, such as for the purpose of assessing percent comple 0113. The term “percent (%) identity,” as used herein, mentarity, can be measured by any Suitable alignment algo refers to the percentage of amino acid (or nucleic acid) rithm, including but not limited to the Needleman-Wunsch residues of a candidate sequence that are identical to the algorithm (see e.g. the EMBOSS Needle aligner available at amino acid (or nucleic acid) residues of a reference sequence www.ebi.ac.uk/Tools/psa/emboss needle/nucleotide.html, after aligning the sequences and introducing gaps, if neces optionally with default settings), the BLAST algorithm (see sary, to achieve the maximum percent identity (i.e., gaps can e.g. the BLAST alignment tool available at blast.ncbi.nlm. be introduced in one or both of the candidate and reference nih.gov/Blast.cgi, optionally with default settings), or the sequences for optimal alignment and non-homologous Smith-Waterman algorithm (see e.g. the EMBOSS Water sequences can be disregarded for comparison purposes). aligner available at www.ebi.ac.uk/Tools/psafemboss wa Alignment, for purposes of determining percent identity, can ter/nucleotide.html, optionally with default settings). Opti be achieved in various ways that are within the skill in the mal alignment can be assessed using any Suitable parameters art, for instance, using publicly available computer software of a chosen algorithm, including default parameters. such as BLAST, ALIGN, or Megalign (DNASTAR) soft 0110 Complementarity can be perfect or substantial/ ware. Percent identity of two sequences can be calculated by sufficient. Perfect complementarity between two nucleic aligning a test sequence with a comparison sequence using acids can mean that the two nucleic acids can form a duplex BLAST, determining the number of amino acids or nucleo in which every base in the duplex is bonded to a comple tides in the aligned test sequence that are identical to amino mentary base by Watson-Crick pairing. Substantial or suf acids or nucleotides in the same position of the comparison ficient complementary can mean that a sequence in one sequence, and dividing the number of identical amino acids Strand is not completely and/or perfectly complementary to or nucleotides by the number of amino acids or nucleotides a sequence in an opposing Strand, but that sufficient bonding in the comparison sequence. occurs between bases on the two strands to form a stable 0114. The term “gene modulating polypeptide' or hybrid complex in set of hybridization conditions (e.g., salt “GMP' as used herein, refers to a polypeptide comprising at concentration and temperature). Such conditions can be least an actuator moiety capable of regulating expression or predicted by using the sequences and standard mathematical activity of a gene and/or editing a nucleic acid sequence. A calculations to predict the Tm of hybridized strands, or by GMP can comprise additional peptide sequences which are empirical determination of Tm by using routine methods. not involved in modulating gene expression, for example 0111. The terms “peptide,” “polypeptide,” and “protein' cleavage recognition sites, linker sequences, targeting are used interchangeably herein to refer to a polymer of at Sequences, etc. least two amino acid residues joined by peptide bond(s). 0115 The terms “actuator moiety,” “actuator domain.” This term does not connote a specific length of polymer, nor and gene modulating domain,” as used herein, refers to a US 2017/O 1983O8 A1 Jul. 13, 2017

moiety which can regulate expression or activity of a gene identical, at least about 99% identical, or 100% identical, to and/or edit a nucleic acid sequence, whether exogenous or a wild type exemplary crRNA sequence (e.g., a crRNA from endogenous. An actuator moiety can regulate expression of S. pyogenes) over a stretch of at least 6 contiguous nucleo a gene at the transcription level and/or the translation level. tides An actuator moiety can regulate gene expression at the 0118. The term “tracrRNA, as used herein, can generally transcription level, for example, by regulating the produc refer to a nucleic acid with at least about 5%, 10%, 20%, tion of mRNA from DNA, such as chromosomal DNA or 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence cDNA. In some embodiments, an actuator moiety recruits at identity and/or sequence similarity to a wild type exemplary least one transcription factor that binds to a specific DNA tracrRNA sequence (e.g., a tracrRNA from S. pyogenes). sequence, thereby controlling the rate of transcription of tracrRNA can refer to a nucleic acid with at most about 5%, genetic information from DNA to mRNA. An actuator 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% moiety can itself bind to DNA and regulate transcription by sequence identity and/or sequence similarity to a wild type physical obstruction, for example preventing proteins such exemplary tracrRNA sequence (e.g., a tracrRNA from S. as RNA polymerase and other associated proteins from pyogenes). tracrRNA can refer to a modified form of a assembling on a DNA template. An actuator moiety can tracrRNA that can comprise a nucleotide change Such as a regulate expression of a gene at the translation level, for deletion, insertion, or Substitution, variant, mutation, or example, by regulating the production of protein from chimera. A tracrRNA can refer to a nucleic acid that can be mRNA template. In some embodiments, an actuator moiety at least about 60% identical to a wild type exemplary regulates gene expression by affecting the stability of an tracrRNA (e.g., a tracrRNA from S. pyogenes) sequence mRNA transcript. In some embodiments, an actuator moiety over a stretch of at least 6 contiguous nucleotides. For regulates expression of a gene by editing a nucleic acid example, a tracrRNA sequence can be at least about 60% sequence (e.g., a region of a genome). In some embodi identical, at least about 65% identical, at least about 70% ments, an actuator moiety regulates expression of a gene by identical, at least about 75% identical, at least about 80% editing an mRNA template. Editing a nucleic acid sequence identical, at least about 85% identical, at least about 90% can, in Some cases, alter the underlying template for gene identical, at least about 95% identical, at least about 98% expression. identical, at least about 99% identical, or 100% identical, to 0116. A Cas protein referred to herein can be a type of a wild type exemplary tracrRNA (e.g., a tracrRNA from S. protein or polypeptide. A Cas protein can refer to a nuclease. pyogenes) sequence over a stretch of at least 6 contiguous A Cas protein can refer to an endoribonuclease. A Cas nucleotides. protein can refer to any modified (e.g., shortened, mutated, lengthened) polypeptide sequence or homologue of the Cas 0119) As used herein, a guide nucleic acid can refer to a nucleic acid that can hybridize to another nucleic acid. A protein. A Cas protein can be codon optimized. A Cas protein guide nucleic acid can be RNA. A guide nucleic acid can be can be a codon-optimized homologue of a Cas protein. A DNA. The guide nucleic acid can be programmed to bind to Cas protein can be enzymatically inactive, partially active, a sequence of nucleic acid site-specifically. The nucleic acid constitutively active, fully active, inducible active and/or to be targeted, or the target nucleic acid, can comprise more active, (e.g. more than the wild type homologue of the nucleotides. The guide nucleic acid can comprise nucleo protein or polypeptide.). A Cas protein can be Cas9. A Cas tides. A portion of the target nucleic acid can be comple protein can be Cpf1. A Cas protein can be C2c2. A Cas mentary to a portion of the guide nucleic acid. The strand of protein (e.g., variant, mutated, enzymatically inactive and/or a double-stranded target polynucleotide that is complemen conditionally enzymatically inactive site-directed polypep tary to and hybridizes with the guide nucleic acid can be tide) can bind to a target nucleic acid. The Cas protein (e.g., called the complementary strand. The strand of the double variant, mutated, enzymatically inactive and/or condition Stranded target polynucleotide that is complementary to the ally enzymatically inactive endoribonuclease) can bind to a complementary strand, and therefore may not be comple target RNA or DNA. mentary to the guide nucleic acid can be called noncomple 0117 The term “crRNA, as used herein, can generally mentary strand. A guide nucleic acid can comprise a poly refer to a nucleic acid with at least about 5%, 10%, 20%, nucleotide chain and can be called a “single guide nucleic 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence acid'. A guide nucleic acid can comprise two polynucleotide identity and/or sequence similarity to a wild type exemplary chains and can be called a “double guide nucleic acid”. If not crRNA (e.g., a crRNA from S. pyogenes). crRNA can otherwise specified, the term “guide nucleic acid can be generally refer to a nucleic acid with at most about 5%, 10%, inclusive, referring to both single guide nucleic acids and 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% double guide nucleic acids. sequence identity and/or sequence similarity to a wild type exemplary crRNA (e.g., a crRNA from S. pyogenes). crRNA 0.120. A guide nucleic acid can comprise a segment that can refer to a modified form of a crRNA that can comprise can be referred to as a “nucleic acid-targeting segment” or a nucleotide change Such as a deletion, insertion, or Substi a “nucleic acid-targeting sequence. A nucleic acid-targeting tution, variant, mutation, or chimera. A crRNA can be a nucleic acid can comprise a segment that can be referred to nucleic acid having at least about 60% identical to a wild as a “protein binding segment’ or “protein binding type exemplary crRNA (e.g., a crRNA from S. pyogenes) sequence' or "Cas protein binding segment'. sequence over a stretch of at least 6 contiguous nucleotides. I0121 The term “cleavage recognition site, as used For example, a crRNA sequence can be at least about 60% herein, with reference to peptides, refers to a site of a peptide identical, at least about 65% identical, at least about 70% at which a chemical bond, such as a peptide bond or disulfide identical, at least about 75% identical, at least about 80% bond, can be cleaved. Cleavage can be achieved by various identical, at least about 85% identical, at least about 90% methods. Cleavage of peptide bonds can be facilitated, for identical, at least about 95% identical, at least about 98% example, by an enzyme such as a protease or by protein US 2017/O 1983O8 A1 Jul. 13, 2017 splicing (e.g., inteins). Cleavage of a disulfide bond can be activity (e.g., enzymatic activity, methyltransferase activity, facilitated, for example, by an enzyme Such as an oxi acetyltransferase activity, kinase activity, ubiquitinating doreductase. activity, etc.) that can also be exhibited by the nucleic acid 0122) The term “targeting sequence,” as used herein, and/or polypeptide sequence to which the non-native refers to a nucleotide sequence and the corresponding amino sequence is fused. A non-native nucleic acid or polypeptide acid sequence which encodes a targeting polypeptide which sequence may be linked to a naturally-occurring nucleic acid mediates the localization (or retention) of a protein to a or polypeptide sequence (or a variant thereof) by genetic Sub-cellular location, e.g., plasma membrane or membrane engineering to generate a chimeric nucleic acid and/or of a given organelle, nucleus, cytosol, mitochondria, endo polypeptide sequence encoding a chimeric nucleic acid plasmic reticulum (ER), Golgi, chloroplast, apoplast, per and/or polypeptide. oxisome or other organelle. For example, a targeting I0126. As used herein, “treating or “treatment” refers to sequence can direct a protein (e.g., a receptor polypeptide or any one of the following: ameliorating one or more symp an adaptor polypeptide) to a nucleus utilizing a nuclear toms of disease, e.g., cancer; preventing the manifestation of localization signal (NLS); outside of a nucleus of a cell, for Such symptoms before they occur, slowing down or com example to the cytoplasm, utilizing a nuclear export signal pletely preventing the progression of the disease (as may be (NES); mitochondria utilizing a mitochondrial targeting evident by longer periods between reoccurrence episodes, signal; the endoplasmic reticulum (ER) utilizing an ER slowing down or prevention of the deterioration of Symp retention signal; a peroxisome utilizing a peroxisomal tar toms, etc.); enhancing the onset of a remission period; geting signal; plasma membrane utilizing a membrane local slowing down the irreversible damage caused in the pro ization signal; or combinations thereof. gressive-chronic stage of the disease (both in the primary 0123. As used herein, “fusion' can refer to a protein and secondary stages); delaying the onset of said progressive and/or nucleic acid comprising one or more non-native stage, or any combination thereof. sequences (e.g., moieties). A fusion can comprise one or I0127. As used herein, “administer,” “administering.” more of the same non-native sequences. A fusion can “administration,” and derivatives thereof refer to the meth comprise one or more of different non-native sequences. A ods that may be used to enable delivery of agents or fusion can be a chimera. A fusion can comprise a nucleic compositions to the desired site of biological action. These acid affinity tag. A fusion can comprise a barcode. A fusion methods include, but are not limited to parenteral adminis can comprise a peptide affinity tag. A fusion can provide for tration (e.g., intravenous, Subcutaneous, intraperitoneal, subcellular localization of the site-directed polypeptide (e.g., intramuscular, intravascular, intrathecal, intranasal, intravit a nuclear localization signal (NLS) for targeting to the real, infusion and local injection), transmucosal injection, nucleus, a mitochondrial localization signal for targeting to oral administration, administration as a Suppository, and the mitochondria, a chloroplast localization signal for tar topical administration. Administration is by any route, geting to a chloroplast, an endoplasmic reticulum (ER) including parenteral. Parenteral administration includes, retention signal, and the like). A fusion can provide a e.g., intravenous, intramuscular, intra-arteriole, intradermal, non-native sequence (e.g., affinity tag) that can be used to Subcutaneous, intraperitoneal, intraventricular, and intracra track or purify. A fusion can be a small molecule Such as nial. Other modes of delivery include, but are not limited to, biotin or a dye such as alexa fluor dyes, Cyanine3 dye, the use of liposomal formulations, intravenous infusion, Cyanine5 dye. transplantation, etc. One skilled in the art will know of 0.124. A fusion can refer to any protein with a functional additional methods for administering a therapeutically effec effect. For example, a fusion protein can comprise methyl tive amount of a composition of the present disclosure for transferase activity, demethylase activity, dismutase activity, preventing or relieving one or more symptoms associated alkylation activity, depurination activity, oxidation activity, with a disease. pyrimidine dimer forming activity, integrase activity, trans I0128 Disclosed herein are systems, methods, and com posase activity, recombinase activity, polymerase activity, positions for regulating expression of a target polynucleotide ligase activity, helicase activity, photolyase activity or gly in a cell. In an aspect, the present disclosure provides a cosylase activity, acetyltransferase activity, deacetylase system for regulating expression of a target polynucleotide activity, kinase activity, phosphatase activity, ubiquitin in a cell. An exemplary system comprises (a) a chimeric ligase activity, deubiquitinating activity, adenylation activ receptor polypeptide that is modified upon binding an anti ity, deadenylation activity, SUMOylating activity, deSU gen, wherein receptor modification comprises a conforma MOylating activity, ribosylation activity, deribosylation tional change or chemical modification, (b) a chimeric activity, myristoylation activity, remodelling activity, pro adaptor polypeptide that binds the receptor in response to the tease activity, oxidoreductase activity, transferase activity, receptor modification, (c) a gene modulating polypeptide hydrolase activity, lyase activity, isomerase activity, Syn (GMP) comprising an actuator moiety linked to a cleavage thase activity, synthetase activity, or demyristoylation activ recognition site, wherein upon cleavage of the cleavage ity. An effector protein can modify a genomic locus. A fusion recognition site, the actuator moiety is activated to complex protein can be a fusion in a Cas protein. An fusion protein with a target polynucleotide, and (d) a cleavage moiety that can be a non-native sequence in a Cas protein. cleaves the cleavage recognition site when in proximity to 0125. As used herein, “non-native' can refer to a nucleic the cleavage recognition site. The chimeric receptor poly acid or polypeptide sequence that is not found in a native peptide, chimeric adaptor polypeptide, gene modulating nucleic acid or protein. Non-native can refer to affinity tags. polypeptide (GMP), and cleavage moiety of a subject sys Non-native can refer to fusions. Non-native can refer to a tem can be arranged in a variety of configurations. Exem naturally occurring nucleic acid or polypeptide sequence plary, non-limiting configurations are described herein. In that comprises mutations, insertions and/or deletions. A some embodiments, the GMP forms a portion of an intrac non-native sequence may exhibit and/or encode for an ellular region of the chimeric receptor polypeptide, and the US 2017/O 1983O8 A1 Jul. 13, 2017 cleavage moiety forms a portion of the chimeric adaptor changes of a chimeric receptor polypeptide can expose one polypeptide. In some embodiments, the GMP forms a por or more regions of the receptor which was previously not tion of the chimeric adaptor polypeptide, and the cleavage exposed, and the exposed region can recruit and/or bind moiety forms a portion of an intracellular region of the signaling protein(s). Chemical modifications on a receptor, chimeric receptor polypeptide. In some embodiments, the for example phosphorylation and/or dephosphorylation cleavage moiety is complexed with a second adaptor poly (e.g., at tyrosine, serine, threonine, and/or any other Suitable peptide that binds the chimeric receptor polypeptide in amino acid residue), can also recruit signaling proteins response to the receptor modification, and the GMP forms a involved in regulating intracellular processes. Signaling portion of the chimeric adaptor polypeptide. proteins can bind directly to a receptor or indirectly to a 0129. In an exemplary configuration, the GMP forms a receptor, for example as part of a larger complex. portion of an intracellular region of the chimeric receptor 0.132. In some embodiments, the chimeric receptor poly polypeptide, and the cleavage moiety forms a portion of the peptide is a transmembrane receptor. An exemplary trans chimeric adaptor polypeptide. A chimeric receptor polypep membrane receptor is shown in FIG. 2. A transmembrane tide of an exemplary configuration can comprise (a) an receptor can be embedded in a cell membrane and have at antigen interacting domain, and (b) a gene modulating least an extracellular region 201, a region spanning a mem polypeptide (GMP) comprising an actuator moiety linked to brane 202 Such as a plasma membrane, and an intracellular a cleavage recognition site. FIG. 1 shows an exemplary region 203. The antigen interacting domain can form a chimeric receptor polypeptide. The receptor comprises an portion of the extracellular region, and the GMP can form a antigen interacting domain 101 and a gene modulating portion of the intracellular region. Membrane receptors can polypeptide (GMP) 102. The GMP 102 can comprise an detect at least one signal. Such as a small molecule, ion, or actuator moiety 102a linked to a cleavage recognition site protein, from the Surrounding environment (e.g., extracel 102b. lular and/or intracellular environment) and can initiate a 0130. In some embodiments, (i) the chimeric receptor cellular response via at least one signaling cascade involving polypeptide is modified in response to antigen binding, (ii) additional proteins and signaling molecules. Some receptors the cleavage recognition site is cleaved by a cleavage moiety can translocate from one region of a cell to another, for in response to modification of the chimeric receptor poly example from the plasma membrane or cytoplasm to the peptide, (iii) the actuator moiety complexes with a target nucleus and vice versa. Such translocation can be condi polynucleotide after being cleaved from the chimeric recep tional upon ligand binding to the receptor. Examples of tor polypeptide at the cleavage recognition site, and (iv) the membrane receptors include, but are not limited to. Notch chimeric receptor polypeptide does not comprise SEQ ID receptors; G-protein coupled receptors (GPCRs); integrin receptors; cadherin receptors; catalytic receptors including NO: 39. receptors possessing enzymatic activity and receptors which, rather than possessing intrinsic enzymatic activity, SEO ID NO: 39 act by Stimulating non-covalently associated enzymes (e.g., ILDYSFTGGAGRDIPPPOIEEACELPECOWDAGNKVCNLOCNNHACGWDG kinases); death receptors such as members of the tumor necrosis factor receptor (TNFR) superfamily; and immune GDCSLNFNDPWKNCTOSLOCWKYFSDGHCDSOCNSAGCLFDGFDCOLTEG receptors. OCNPLYDOYCKDHFSDGHCDOGCNSAECEWDGLDCAEHVPERLAAGTLVL I0133. In some embodiments, a chimeric receptor poly peptide comprises a Notch receptor, or any derivative, WWLLPPDOLRNNSFHFLRELSHVLHTNVVFKRDAOGOOMIFPYYGHEEEL variant or fragment thereof. Notch receptors are transmem RKHPIKRSTVGWATSSLLPGTSGGRORRELDPMDIRGSIWYLEIDNROCV brane proteins that mediate cell-cell contact signaling and play a central role in development and other aspects of QSSSOCFOSATDWAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHL cell-to-cell communication, e.g. communication between two contacting cells (receiver cell and sending cell). Notch MYWAAAAFWLLFFWGCGWLLSRKRRR receptors expressed in a receiver cell recognize their ligands 0131. A chimeric receptor polypeptide of a subject sys (the delta family of proteins), expressed on a sending cell. tem can comprise an endogenous receptor, or any derivative, The engagement of notch and delta on these contacting cells variant or fragment thereof. A chimeric receptor polypeptide leads to two-step proteolysis of the notch receptor that can bind specifically to at least one antigen (e.g., at least one ultimately causes the release of the intracellular portion of ligand), for example via an antigen interacting domain (also the receptor from the membrane into the cytoplasm. referred to herein as an “extracellular sensor domain'). A I0134. In some embodiments, a chimeric receptor poly chimeric receptor polypeptide can, in response to ligand peptide comprises at least an extracellular region (e.g., binding, undergo a modification Such as a conformational ligand binding domain) of a Notch receptor, or any deriva change and/or chemical modification. Such modification(s) tive, variant or fragment thereof. In some embodiments, a can recruit to the receptor binding partners (e.g., partners chimeric receptor polypeptide comprises at least a mem Such as proteins) including, but not limited to, signaling brane spanning region of a Notch, or any derivative, variant proteins involved in signaling events and various cellular or fragment thereof. In some embodiments, a chimeric processes. Signaling proteins, for example, can be involved receptor polypeptide comprises at least an intracellular in regulating (e.g., activating and/or de-activating) a cellular region (e.g., cytoplasmic domain) of a Notch, or any deriva response Such as programmed changes in gene expression tive, variant or fragment thereof. A chimeric receptor poly via translational regulation; transcriptional regulation; and peptide comprising a Notch, or any derivative, variant or epigenetic modification including the regulation of methyl fragment thereof, can recruit a binding partner. In some ation, acetylation, phosphorylation, ubiquitylation, Sumoy embodiments, ligand binding to a chimeric receptor com lation, ribosylation, and citrullination. Conformational prising a Notch, or any derivative, variant or fragment US 2017/O 1983O8 A1 Jul. 13, 2017

thereof, results in a conformational change, chemical modi B receptors; neuropeptide Y receptors; neurotensin recep fication, or combination thereof, which recruits a binding tors; opioid receptors; orexin receptors; oxoglutarate recep partner to the receptor. tor; P2Y receptors; parathyroid hormone receptors; platelet 0135) In some embodiments, a chimeric receptor poly activating factor receptor, prokineticin receptors; prolactin peptide comprises a Notch, or any derivative, variant or releasing peptide receptor, prostanoid receptors; proteinase fragment thereof, selected from Notch1, Notch2, Notch3. activated receptors; QRFP receptor; relaxin family peptide and Notch4 or any homolog thereof. receptors; somatostatin receptors; Succinate receptor; tachy 0136. In some embodiments, a chimeric receptor poly kinin receptors; thyrotropin-releasing hormone receptors; peptide comprises a G-protein coupled receptor (GPCR), or trace amine receptor, urotensin receptor; vasopressin and any derivative, variant or fragment thereof. GPCRs are oxytocin receptors; VIP and PACAP receptors. generally characterized by seven membrane-spanning a heli 0.139. In some embodiments, a chimeric receptor poly ces and can be arranged in a tertiary structure resembling a peptide comprises a GPCR selected from the group consist barrel, with the seven transmembrane helices forming a ing of 5-hydroxytryptamine (serotonin) receptor 1A cavity within the plasma membrane that serves as a ligand (HTR1A), 5-hydroxytryptamine (serotonin) receptor 1B binding domain. Ligands can also bind elsewhere to a (HTR1B), 5-hydroxytryptamine (serotonin) receptor 1D GPCR, for example to the extracellular loops and/or the (HTR1D), 5-hydroxytryptamine (serotonin) receptor 1E N-terminal tail. Ligand binding can activate an associated G (HTR1E), 5-hydroxytryptamine (serotonin) receptor 1F protein, which then functions in various signaling pathways. (HTR1F), 5-hydroxytryptamine (serotonin) receptor 2A To de-activate this signaling, a GPCR can first be chemically (HTR2A), 5-hydroxytryptamine (serotonin) receptor 2B modified by phosphorylation. Phosphorylation can then (HTR2B), 5-hydroxytryptamine (serotonin) receptor 2C recruit co-adaptor proteins (e.g., arrestin proteins) for addi (HTR2C), 5-hydroxytryptamine (serotonin) receptor 4 tional signaling. (HTR4), 5-hydroxytryptamine (serotonin) receptor 5A 0.137 In some embodiments, a chimeric receptor poly (HTR5A), 5-hydroxytryptamine (serotonin) receptor 5B peptide comprises at least an extracellular region (e.g., (HTR5BP), 5-hydroxytryptamine (serotonin) receptor 6 ligand binding domain) of a GPCR, or any derivative, (HTR6), 5-hydroxytryptamine (serotonin) receptor 7. variant or fragment thereof. In some embodiments, a chi adenylate cyclase-coupled (HTR7), cholinergic receptor, meric receptor polypeptide comprises at least a membrane muscarinic 1 (CHRM1), cholinergic receptor, muscarinic 2 spanning region of a GPCR, or any derivative, variant or (CHRM2), cholinergic receptor, muscarinic 3 (CHRM3), fragment thereof. In some embodiments, a chimeric receptor cholinergic receptor, muscarinic 4 (CHRM4), cholinergic polypeptide comprises at least an intracellular region (e.g., receptor, muscarinic 5 (CHRM5), adenosine A1 receptor cytoplasmic domain) of a GPCR, or any derivative, variant (ADORA1), adenosine A2a receptor (ADORA2A), adenos or fragment thereof. A chimeric receptor polypeptide com ine A2b receptor (ADORA2B), adenosine A3 receptor prising a GPCR, or any derivative, variant or fragment (ADORA3), adhesion protein-coupled receptor A1 thereof, can recruit a binding partner. In some embodiments, (ADGRA1), adhesion protein-coupled receptor A2 ligand binding to a chimeric receptor comprising a GPCR, (ADGRA2), adhesion protein-coupled receptor A3 or any derivative, variant or fragment thereof, results in a (ADGRA3), adhesion protein-coupled receptor B1 conformational change, chemical modification, or combina (ADGRB1), adhesion protein-coupled receptor B2 tion thereof, which recruits a binding partner to the receptor. (ADGRB2), adhesion protein-coupled receptor B3 0.138. In some embodiments, a chimeric receptor poly (ADGRB3), cadherin EGF LAG seven-pass G-type receptor peptide comprises a GPCR, or any derivative, variant or 1 (CELSR1), cadherin EGF LAG seven-pass G-type recep fragment thereof, selected from Class A Orphans; Class B tor 2 (CELSR2), cadherin EGF LAG seven-pass G-type Orphans; Class C Orphans; taste receptors, type 1; taste receptor 3 (CELSR3), adhesion G protein-coupled receptor receptors, type 2: 5-hydroxytryptamine receptors; acetyl D1 (ADGRD1), adhesion G protein-coupled receptor D2 choline receptors (muscarinic); adenosine receptors; adhe (ADGRD2), adhesion G protein-coupled receptor E1 sion class GPCRs, adrenoceptors; angiotensin receptors; (ADGRE1), adhesion G protein-coupled receptor E2 apelin receptor; bile acid receptor; bombesin receptors; (ADGRE2), adhesion G protein-coupled receptor E3 bradykinin receptors; calcitonin receptors; calcium-sensing (ADGRE3), adhesion G protein-coupled receptor E4 receptors; cannabinoid receptors; chemerin receptor, (ADGRE4P), adhesion G protein-coupled receptor E5 chemokine receptors; cholecystokinin receptors; class (ADGRE5), adhesion protein-coupled receptor F1 Frizzled GPCRs (e.g., Wnt receptors); complement peptide (ADGRF1), adhesion protein-coupled receptor F2 receptors; corticotropin-releasing factor receptors; dop (ADGRF2), adhesion protein-coupled receptor F3 amine receptors; endothelin receptors; G protein-coupled (ADGRF3), adhesion protein-coupled receptor F4 estrogen receptor, formylpeptide receptors; free fatty acid (ADGRF4), adhesion protein-coupled receptor F5 receptors; GABAB receptors; galanin receptors; ghrelin (ADGRF5), adhesion protein-coupled receptor G1 receptor; glucagon receptor family; glycoprotein hormone (ADGRG1), adhesion protein-coupled receptor G2 receptors; gonadotrophin-releasing hormone receptors; (ADGRG2), adhesion protein-coupled receptor G3 GPR18, GPR55 and GPR119; histamine receptors; hydroxy (ADGRG3), adhesion protein-coupled receptor G4 carboxylic acid receptors; kisspeptin receptor, leukotriene (ADGRG4), adhesion protein-coupled receptor G5 receptors; lysophospholipid (LPA) receptors; lysophospho (ADGRG5), adhesion protein-coupled receptor G6 lipid (S1P) receptors; melanin-concentrating hormone (ADGRG6), adhesion protein-coupled receptor G7 receptors; melanocortin receptors; melatonin receptors; (ADGRG7), adhesion protein-coupled receptor L1 metabotropic glutamate receptors; motilin receptor; neuro (ADGRL1), adhesion protein-coupled receptor L2 medin U receptors; neuropeptide FF/neuropeptide AF recep (ADGRL2), adhesion protein-coupled receptor L3 tors; neuropeptide S receptor; neuropeptide W/neuropeptide (ADGRL3), adhesion protein-coupled receptor L4 US 2017/O 1983O8 A1 Jul. 13, 2017

(ADGRL4), adhesion G protein-coupled receptor V1 tein-coupled receptor 78 (GPR78), G protein-coupled recep (ADGRV1), adrenoceptor alpha 1A (ADRA1A), adrenocep tor 79 (GPR79), G protein-coupled receptor 82 (GPR82), G tor alpha 1B (ADRA1B), adrenoceptor alpha 1D protein-coupled receptor 83 (GPR83), G protein-coupled (ADRA1D), adrenoceptor alpha 2A (ADRA2A), adrenocep receptor 84 (GPR84), G protein-coupled receptor 85 tor alpha 2B (ADRA2B), adrenoceptor alpha 2C (GPR85), G protein-coupled receptor 87 (GPR87), G pro (ADRA2C), adrenoceptor beta 1 (ADRB1), adrenoceptor tein-coupled receptor 88 (GPR88), G protein-coupled recep beta 2 (ADRB2), adrenoceptor beta 3 (ADRB3), angiotensin tor 101 (GPR101), G protein-coupled receptor 119 II receptor type 1 (AGTR1), angiotensin II receptor type 2 (GPR119), G protein-coupled receptor 132 (GPR132), G (AGTR2), apelin receptor (APLNR), G protein-coupled bile protein-coupled receptor 135 (GPR135), G protein-coupled acid receptor 1 (GPBAR1), neuromedin B receptor receptor 139 (GPR139), G protein-coupled receptor 141 (NMBR), gastrin releasing peptide receptor (GRPR), bomb (GPR141), G protein-coupled receptor 142 (GPR142), G esin like receptor 3 (BRS3), bradykinin receptor B1 (BD protein-coupled receptor 146 (GPR146), G protein-coupled KRB1), bradykinin receptor B2 (BDKRB2), calcitonin receptor 148 (GPR148), G protein-coupled receptor 149 receptor (CALCR), calcitonin receptor like receptor (CAL (GPR149), G protein-coupled receptor 150 (GPR150), G CRL), calcium sensing receptor (CASR), G protein-coupled protein-coupled receptor 151 (GPR151), G protein-coupled receptor, class C (GPRC6A), cannabinoid receptor 1 (brain) receptor 152 (GPR152), G protein-coupled receptor 153 (CNR1), cannabinoid receptor 2 (CNR2), chemerin (GPR153), G protein-coupled receptor 160 (GPR160), G chemokine-like receptor 1 (CMKLR1), chemokine (C-C protein-coupled receptor 161 (GPR161), G protein-coupled motif) receptor 1 (CCR1), chemokine (C-C motif) receptor receptor 162 (GPR162), G protein-coupled receptor 171 2 (CCR2), chemokine (C-C motif) receptor 3 (CCR3), (GPR171), G protein-coupled receptor 173 (GPR173), G chemokine (C-C motif) receptor 4 (CCR4), chemokine (C-C protein-coupled receptor 174 (GPR174), G protein-coupled motif) receptor 5 (gene/pseudogene) (CCRS), chemokine receptor 176 (GPR176), G protein-coupled receptor 182 (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) (GPR182), G protein-coupled receptor 183 (GPR183), leu receptor 7 (CCR7), chemokine (C-C motif) receptor 8 cine-rich repeat containing G protein-coupled receptor 4 (CCR8), chemokine (C-C motif) receptor 9 (CCR9), (LGR4), leucine-rich repeat containing G protein-coupled chemokine (C-C motif) receptor 10 (CCR10), chemokine receptor 5 (LGR5), leucine-rich repeat containing G protein (C-X-C motif) receptor 1 (CXCR1), chemokine (C-X-C coupled receptor 6 (LGR6), MASI proto-oncogene (MAS motif) receptor 2 (CXCR2), chemokine (C-X-C motif) 1), MASI proto-oncogene like (MAS1L), MAS related GPR receptor 3 (CXCR3), chemokine (C-X-C motif) receptor 4 family member D (MRGPRD), MAS related GPR family (CXCR4), chemokine (C-X-C motif) receptor 5 (CXCR5), member E (MRGPRE), MAS related GPR family member F chemokine (C-X-C motif) receptor 6 (CXCR6), chemokine (MRGPRF), MAS related GPR family member G (MRG (C-X3-C motif) receptor 1 (CX3CR1), chemokine (C motif) PRG), MAS related GPR family member X1 (MRGPRX1), receptor 1 (XCR1), atypical chemokine receptor 1 (Duffy MAS related GPR family member X2 (MRGPRX2), MAS blood group) (ACKR1), atypical chemokine receptor 2 related GPR family member X3 (MRGPRX3), MAS related (ACKR2), atypical chemokine receptor 3 (ACKR3), atypi GPR family member X4 (MRGPRX4), opsin 3 (OPN3), cal chemokine receptor 4 (ACKR4), chemokine (C-C motif) opsin 4 (OPN4), opsin 5 (OPNS), purinergic receptor P2Y receptor-like 2 (CCRL2), cholecystokinin A receptor (P2RY8), purinergic receptor P2Y (P2RY10), trace amine (CCKAR), cholecystokinin B receptor (CCKBR), G pro associated receptor 2 (TAAR2), trace amine associated tein-coupled receptor 1 (GPR1), bombesin like receptor 3 receptor 3 (gene/pseudogene) (TAAR3), trace amine asso (BRS3), G protein-coupled receptor 3 (GPR3), G protein ciated receptor 4 (TAAR4P), trace amine associated receptor coupled receptor 4 (GPR4), G protein-coupled receptor 6 5 (TAAR5), trace amine associated receptor 6 (TAAR6), (GPR6), G protein-coupled receptor 12 (GPR12), G protein trace amine associated receptor 8 (TAAR8), trace amine coupled receptor 15 (GPR15), G protein-coupled receptor associated receptor 9 (gene/pseudogene) (TAAR9), G pro 17 (GPR17), G protein-coupled receptor 18 (GPR18), G tein-coupled receptor 156 (GPR156), G protein-coupled protein-coupled receptor 19 (GPR19), G protein-coupled receptor 158 (GPR158), G protein-coupled receptor 179 receptor 20 (GPR20), G protein-coupled receptor 21 (GPR179), G protein-coupled receptor, class C (GPRC5A), (GPR21), G protein-coupled receptor 22 (GPR22), G pro G protein-coupled receptor, class C (GPRC5B), G protein tein-coupled receptor 25 (GPR25), G protein-coupled recep coupled receptor, class C (GPRC5C), G protein-coupled tor 26 (GPR26), G protein-coupled receptor 27 (GPR27), G receptor, class C (GPRC5D), frizzled class receptor 1 protein-coupled receptor 31 (GPR31), G protein-coupled (FZD1), frizzled class receptor 2 (FZD2), frizzled class receptor 32 (GPR32), G protein-coupled receptor 33 (gene/ receptor 3 (FZD3), frizzled class receptor 4 (FZD4), frizzled pseudogene) (GPR33), G protein-coupled receptor 34 class receptor 5 (FZD5), frizzled class receptor 6 (FZD6), (GPR34), G protein-coupled receptor 35 (GPR35), G pro frizzled class receptor 7 (FZD7), frizzled class receptor 8 tein-coupled receptor 37 (endothelin receptor type B-like) (FZD8), frizzled class receptor 9 (FZD9), frizzled class (GPR37), G protein-coupled receptor 37 like 1 (GPR37L1), receptor 10 (FZD10), smoothened, frizzled class receptor G protein-coupled receptor 39 (GPR39), G protein-coupled (SMO), complement component 3a receptor 1 (C3AR1), receptor 42 (gene/pseudogene) (GPR42), G protein-coupled complement component 5a receptor 1 (C5AR1), comple receptor 45 (GPR45), G protein-coupled receptor 50 ment component 5a receptor 2 (C5AR2), corticotropin (GPR50), G protein-coupled receptor 52 (GPR52), G pro releasing hormone receptor 1 (CRHR1), corticotropin tein-coupled receptor 55 (GPR55), G protein-coupled recep releasing hormone receptor 2 (CRHR2), dopamine receptor tor 61 (GPR61), G protein-coupled receptor 62 (GPR62), G D1 (DRD1), dopamine receptor D2 (DRD2), dopamine protein-coupled receptor 63 (GPR63), G protein-coupled receptor D3 (DRD3), dopamine receptor D4 (DRD4), dop receptor 65 (GPR65), G protein-coupled receptor 68 amine receptor D5 (DRD5), endothelin receptor type A (GPR68), G protein-coupled receptor 75 (GPR75), G pro (EDNRA), endothelin receptor type B (EDNRB), formyl US 2017/O 1983O8 A1 Jul. 13, 2017 peptide receptor 1 (FPR1), formyl peptide receptor 2 (OPRD1), opioid receptor, kappa 1 (OPRK1), opioid recep (FPR2), formyl peptide receptor 3 (FPR3), free fatty acid tor, mu 1 (OPRM1), opiate receptor-like 1 (OPRL1), hypo receptor 1 (FFAR1), free fatty acid receptor 2 (FFAR2), free cretin (orexin) receptor 1 (HCRTR1), hypocretin (orexin) fatty acid receptor 3 (FFAR3), free fatty acid receptor 4 receptor 2 (HCRTR2), G protein-coupled receptor 107 (FFAR4), G protein-coupled receptor 42 (gene/pseudogene) (GPR107), G protein-coupled receptor 137 (GPR137), (GPR42), gamma-aminobutyric acid (GABA) B receptor, 1 olfactory receptor family 51 subfamily E member 1 (GABBR1), gamma-aminobutyric acid (GABA) B receptor, (OR51E1), transmembrane protein, adipocyte associated 1 2 (GABBR2), galanin receptor 1 (GALR1), galanin receptor (TPRA1), G protein-coupled receptor 143 (GPR143), G 2 (GALR2), galanin receptor 3 (GALR3), growth hormone protein-coupled receptor 157 (GPR157), oxoglutarate (al secretagogue receptor (GHSR), growth hormone releasing pha-ketoglutarate) receptor 1 (OXGR1), purinergic receptor hormone receptor (GHRHR), gastric inhibitory polypeptide P2Y (P2RY1), purinergic receptor P2Y (P2RY2), pyrimi receptor (GIPR), glucagon like peptide 1 receptor (GLP1R), dinergic receptor P2Y (P2RY4), pyrimidinergic receptor glucagon-like peptide 2 receptor (GLP2R), glucagon recep P2Y (P2RY6), purinergic receptor P2Y (P2RY11), puriner tor (GCGR), secretin receptor (SCTR), follicle stimulating gic receptor P2Y (P2RY12), purinergic receptor P2Y hormone receptor (FSHR), luteinizing hormone/choriogo (P2RY13), purinergic receptor P2Y (P2RY14), parathyroid nadotropin receptor (LHCGR), thyroid stimulating hormone hormone 1 receptor (PTH1R), parathyroid hormone 2 recep receptor (TSHR), gonadotropin releasing hormone receptor tor (PTH2R), platelet-activating factor receptor (PTAFR), (GNRHR), gonadotropin releasing hormone receptor 2 prokineticin receptor 1 (PROKR1), prokineticin receptor 2 (pseudogene) (GNRHR2), G protein-coupled receptor 18 (PROKR2), prolactin releasing hormone receptor (PRLHR), (GPR18), G protein-coupled receptor 55 (GPR55), G pro prostaglandin D2 receptor (DP) (PTGDR), prostaglandin D2 tein-coupled receptor 119 (GPR119), G protein-coupled receptor 2 (PTGDR2), prostaglandin E receptor 1 (PT estrogen receptor 1 (GPER1), histamine receptor H1 GER1), prostaglandin E receptor 2 (PTGER2), prostaglan (HRH1), histamine receptor H2 (HRH2), histamine receptor din E receptor 3 (PTGER3), prostaglandin E receptor 4 H3 (HRH3), histamine receptor H4 (HRH4), hydroxycar (PTGER4), prostaglandin F receptor (PTGFR), prostaglan boxylic acid receptor 1 (HCAR1), hydroxycarboxylic acid din 12 (prostacyclin) receptor (IP) (PTGIR), thromboxane receptor 2 (HCAR2), hydroxycarboxylic acid receptor 3 A2 receptor (TBXA2R), coagulation factor II thrombin (HCAR3), KISS1 receptor (KISS1R), leukotriene B4 recep receptor (F2R), F2R like trypsin receptor 1 (F2RL1), coagul tor (LTB4R), leukotriene B4 receptor 2 (LTB4R2), cysteinyl lation factor II thrombin receptor like 2 (F2RL2), F2R like leukotriene receptor 1 (CYSLTR1), cysteinyl leukotriene thrombin/trypsin receptor 3 (F2RL3), pyroglutamylated receptor 2 (CYSLTR2), oxoeicosanoid (OXE) receptor 1 RFamide peptide receptor (QRFPR), relaxin/insulin-like (OXER1), formyl peptide receptor 2 (FPR2), lysophospha family peptide receptor 1 (RXFP1), relaxin/insulin-like fam tidic acid receptor 1 (LPAR1), lysophosphatidic acid recep ily peptide receptor 2 (RXFP2), relaxin/insulin-like family tor 2 (LPAR2), lysophosphatidic acid receptor 3 (LPAR3), peptide receptor 3 (RXFP3), relaxin/insulin-like family pep lysophosphatidic acid receptor 4 (LPAR4), lysophosphatidic tide receptor 4 (RXFP4), somatostatin receptor 1 (SSTR1), acid receptor 5 (LPAR5), lysophosphatidic acid receptor 6 somatostatin receptor 2 (SSTR2), somatostatin receptor 3 (LPAR6), sphingosine-1-phosphate receptor 1 (S1PR1), (SSTR3), somatostatin receptor 4 (SSTR4), somatostatin sphingosine-1-phosphate receptor 2 (S1PR2), sphingosine receptor 5 (SSTR5), succinate receptor 1 (SUCNR1), tachy 1-phosphate receptor 3 (S1PR3), sphingosine-1-phosphate kinin receptor 1 (TACR1), tachykinin receptor 2 (TACR2), receptor 4 (S1PR4), sphingosine-1-phosphate receptor 5 tachykinin receptor 3 (TACR3), taste 1 receptor member 1 (S1PR5), melanin concentrating hormone receptor 1 (TAS1R1), taste 1 receptor member 2 (TAS1R2), taste 1 (MCHR1), melanin concentrating hormone receptor 2 receptor member 3 (TAS1R3), taste 2 receptor member 1 (MCHR2), melanocortin 1 receptor (alpha melanocyte (TAS2R1), taste 2 receptor member 3 (TAS2R3), taste 2 stimulating hormone receptor) (MC1R), melanocortin 2 receptor member 4 (TAS2R4), taste 2 receptor member 5 receptor (adrenocorticotropic hormone) (MC2R), melano (TAS2R5), taste 2 receptor member 7 (TAS2R7), taste 2 cortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), receptor member 8 (TAS2R8), taste 2 receptor member 9 melanocortin 5 receptor (MC5R), melatonin receptor 1A (TAS2R9), taste 2 receptor member 10 (TAS2R10), taste 2 (MTNR1A), melatonin receptor 1B (MTNR1B), glutamate receptor member 13 (TAS2R13), taste 2 receptor member 14 receptor, metabotropic 1 (GRM1), glutamate receptor, (TAS2R14), taste 2 receptor member 16 (TAS2R16), taste 2 metabotropic 2 (GRM2), glutamate receptor, metabotropic 3 receptor member 19 (TAS2R 19), taste 2 receptor member 20 (GRM3), glutamate receptor, metabotropic 4 (GRM4), glu (TAS2R20), taste 2 receptor member 30 (TAS2R30), taste 2 tamate receptor, metabotropic 5 (GRM5), glutamate recep receptor member 31 (TAS2R31), taste 2 receptor member 38 tor, metabotropic 6 (GRM6), glutamate receptor, metabo (TAS2R38), taste 2 receptor member 39 (TAS2R39), taste 2 tropic 7 (GRM7), glutamate receptor, metabotropic 8 receptor member 40 (TAS2R40), taste 2 receptor member 41 (GRM8), motilin receptor (MLNR), neuromedin U receptor (TAS2R41), taste 2 receptor member 42 (TAS2R42), taste 2 1 (NMUR1), neuromedin U receptor 2 (NMUR2), neuro receptor member 43 (TAS2R43), taste 2 receptor member 45 peptide FF receptor 1 (NPFFR1), neuropeptide FF receptor (TAS2R45), taste 2 receptor member 46 (TAS2R46), taste 2 2 (NPFFR2), neuropeptide S receptor 1 (NPSR1), neuro receptor member 50 (TAS2R50), taste 2 receptor member 60 peptides B/W receptor 1 (NPBWR1), neuropeptides B/W (TAS2R60), thyrotropin-releasing hormone receptor receptor 2 (NPBWR2), neuropeptide Y receptor Y1 (TRHR), trace amine associated receptor 1 (TAAR1), uro (NPY1R), neuropeptide Y receptor Y2 (NPY2R), neuropep tensin2 receptor (UTS2R), arginine vasopressin receptor 1A tide Y receptor Y4 (NPY4R), neuropeptide Y receptor Y5 (AVPR1A), arginine vasopressin receptor 1B (AVPR1B), (NPY5R), neuropeptide Y receptor Y6 (pseudogene) arginine vasopressin receptor 2 (AVPR2), oxytocin receptor (NPY6R), neurotensin receptor 1 (high affinity) (NTSR1), (OXTR), adenylate cyclase activating polypeptide 1 (pitu neurotensin receptor 2 (NTSR2), opioid receptor, delta 1 itary) receptor type I (ADCYAP1R1), vasoactive intestinal US 2017/O 1983O8 A1 Jul. 13, 2017

peptide receptor 1 (VIPR1), vasoactive intestinal peptide relaxin, relaxin-1, relaxin-3, resolvin D1, resolvin E1, receptor 2 (VIPR2), any derivative thereof, any variant RFRP-1, RFRP-3, R-spondins, secretin, serine proteases, thereof, and any fragment thereof. sphingosine 1-phosphate, sphingosylphosphorylcholine, SRIF-14, SRIF-28, Substance P. Succinic acid, thrombin, 0140. A chimeric receptor polypeptide comprising a thromboxane A2, TIP39, T-kinin, TRH, TSH, tyramine, GPCR, or any derivative, variant or fragment thereof, can UDP-glucose, uridine diphosphate, urocortin 1, urocortin 2, bind an antigen comprising any Suitable GPCR ligand, or urocortin 3, urotensin II-related peptide, urotensin-II, vaso any derivative, variant or fragment thereof. Non-limiting pressin, VIP, Wnt, Wnt-1, Wnt-10a, Wnt-10b, Wnt-11, Wnt examples of ligands which can be bound by a GPCR include 16, Wnt-2, Wnt-2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt (-)-adrenaline, (-)-noradrenaline, (lyso)phospholipid 5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt mediators, des-Arg10kallidin, des-Arg9bradykinin, des 9b. XCL1, XCL2, Zn2+, C.-CGRP. C.-ketoglutaric acid, Gln14ghrelin, Hyp3bradykinin, Leuenkephalin, Met C-MSH, C.-neoendorphin, B-alanine, B-CGRP. B-D-hy enkephalin, 12-hydroxyheptadecatrienoic acid, 12R-HETE, droxybutyric acid, B-endorphin, B-MSH, B-neoendorphin, 12S-HETE, 12S-HPETE, 15S-HETE, 17B-estradiol, 20-hy droxy-LTB4, 2-arachidonoylglycerol, 2-oleoyl-LPA, 3-hy B-phenylethylamine, and Y-MSH. droxyoctanoic acid, 5-hydroxytryptamine, 5-oxo-15-HETE, 0.141. In some embodiments, a chimeric receptor poly 5-oxo-ETE, 5-oxo-ETrE, 5-oxo-ODE, 5S-HETE, peptide comprises an integrin receptor, an integrin receptor 5S-HPETE, 7c.,25-dihydroxycholesterol, acetylcholine, Subunit, or any derivative, variant or fragment thereof. ACTH, adenosine diphosphate, adenosine, adrenomedulin Integrin receptors are transmembrane receptors that can 2/intermedin, adrenomedullin, amylin, anandamide, angio function as bridges for cell-cell and cell-extracellular matrix tensin II, angiotensin III, annexin I, apelin receptor early (ECM) interactions. Integrin receptors are generally formed endogenous ligand, apelin-13, apelin-17, apelin-36, aspirin as heterodimers consisting of an a subunit and a B Subunit triggered lipoxin A4, aspirin-triggered resolvin D1, ATP, which associate non-covalently. There exist at least 18 C. beta-defensin 4A, big dynorphin, bovine adrenal medulla Subunits and at least 8 B Subunits. Each subunit generally peptide 8-22, bradykinin, C3a, C5a, Ca2+, calcitonin gene comprises an extracellular region (e.g., ligand binding related peptide, calcitonin, cathepsin G, CCK-33, CCK-4, domain), a region spanning a membrane, and an intracellular CCK-8, CCL1, CCL11, CCL13, CCL14. CCL15, CCL16, region (e.g., cytoplasmic domain). In some embodiments, a CCL17, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, chimeric receptor polypeptide comprises at least an extra CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, cellular region (e.g., ligand binding domain) of an integrin CCL5, CCL7, CCL8, chemerin, chenodeoxycholic acid, subunit (e.g., C. Subunit or f subunit), or any derivative, cholic acid, corticotrophin-releasing hormone, CST-17, variant or fragment thereof. In some embodiments, a chi CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12O, meric receptor polypeptide comprises at least a region CXCL12?, CXCL13, CXCL16, CXCL2, CXCL3, CXCL5, spanning a membrane of an integrin Subunit (e.g., C.Subunit CXCL6, CXCL7, CXCL8, CXCL9, cysteinyl-leukotrienes or B Subunit), or any derivative, variant or fragment thereof. (CySLTs), uracil nucleotides, deoxycholic acid, dihy In some embodiments, a chimeric receptor polypeptide drosphingosine-1-phosphate, dioleoylphosphatidic acid, comprises at least an intracellular region (e.g., cytoplasmic dopamine, dynorphin A, dynorphin A-(1-13), dynorphin domain) of an integrin subunit (e.g., C. Subunit or B subunit), A-(1-8), dynorphin B, endomorphin-1, endothelin-1, or any derivative, variant or fragment thereof. A chimeric endothelin-2, endothelin-3, F2L, Free fatty acids, FSH. receptor polypeptide comprising an integrin subunit, or any GABA, galanin, galanin-like peptide, gastric inhibitory derivative, variant or fragment thereof, can recruit a binding polypeptide, gastrin-17, gastrin-releasing peptide, ghrelin, partner. In some embodiments, ligand binding to a chimeric GHRH, glucagon, glucagon-like peptide 1-(7-36) amide, receptor comprising an integrin subunit, or any derivative, glucagon-like peptide 1-(7-37), glucagon-like peptide 2. variant or fragment thereof, results in a conformational glucagon-like peptide 2-(3-33), GnRH I, GnRH II, GRP change, chemical modification, or combination thereof, (18-27), hCG, histamine, humanin, INSL3, INSL5, kallidin, which recruits a binding partner to the receptor. kisspeptin-10, kisspeptin-13, kisspeptin-14, kisspeptin-54. 0142. In some embodiments, a chimeric receptor poly kynurenic acid, large neuromedin N, large neurotensin, peptide comprises an integrin receptor a subunit, or any L-glutamic acid, LH, lithocholic acid, L-lactic acid, long derivative, variant or fragment thereof, selected from the chain carboxylic acids, LPA, LTB4, LTC4, LTD4, LTE4. group consisting of C.1, C2, C3, C4, C5, C6, C7, C8, C.9. LXA4, Lys-Hyp3-bradykinin, lysophosphatidylinositol, C.10, C. 11, C.V. C.L., C.M., OX, O.D., C.E., and C.IIb. In some lysophosphatidylserine, Medium-chain-length fatty acids, embodiments, a chimeric receptor polypeptide comprises an melanin-concentrating hormone, melatonin, methylcar integrin receptor B Subunit, or any derivative, variant or bamyl PAF, Mg2+, motilin, N-arachidonoylglycine, neuro fragment thereof, selected from the group consisting of 31, kinin A, neurokinin B, neuromedin B, neuromedin N, neu B2, B3, B4, B5, B6, B7, and 38. Chimeric receptor polypep romedin S-33, neuromedin U-25, neuronostatin, tides comprising an O. Subunit, a B subunit, or any derivative, neuropeptide AF, neuropeptide B-23, neuropeptide B-29, variant or fragment thereof, can heterodimerize (e.g., C. neuropeptide FF, neuropeptide S, neuropeptide SF, neuro Subunit dimerizing with a B subunit) to form an integrin peptide W-23, neuropeptide W-30, neuropeptide Y, neuro receptor, or any derivative, variant or fragment thereof. peptide Y-(3-36), neurotensin, nociceptin/orphanin FQ. Non-limiting examples of integrin receptors include an N-oleoylethanolamide, obestatin, octopamine, orexin-A, C.131, C231, C3 B1, C4 B1, C.5 B1, C.6B1, C.7f81, C.8f31, C.931, orexin-B, Oxysterols, oxytocin, PACAP-27, PACAP-38, C.1081, CVB1, OLB1, CMB1, CXR1, CDR1, CIIb?31, CEB1, PAF, pancreatic polypeptide, peptide YY. PGD2, PGE2, C.132. C.232, C3 B2, C4 B2, C.5 B2, C.6B2. C.7f82, C.8f32, C.932, PGF2C, PGI2, PGJ2, PHM, phosphatidylserine, PHV, pro C.10B2, CVB2, C.L.B2, C.M.B2, CXB2, CDB2, C.IIb?32, CEB2. kineticin-1, prokineticin-2, prokineticin-2B, prosaposin, C.1 f3, O2B3, C3 B3, C4 B3, C.5 B3, C.6B3, C.7f83, C8B3, C.9B3, PrRP-20, PrRP-31, PTH, PTHrP. PTHrP-(1-36), QRFP43, C.1083, CVB3, C.L.f33, C.M.B3, CXR3, CDR3, CIIb?33, CEB3, US 2017/O 1983O8 A1 Jul. 13, 2017

C.134, C.234, C3 B4, C4 B4, C.5 B4, C.6B4, C.7f84, C.8f34, C.934, protocadherin, and an unconventional cadherin. In some C. 1034, C.VB4, C.L.f34, CMB4, CXB4, C.DB4, C.IIb?34, CEB4. embodiments, a chimeric receptor polypeptide comprises a C.1?5, O2B5, C3 B5, C4 B5, C.5 B5, C.6B5, O7B5, C8B5, c.985, classical cadherin, or any derivative, variant or fragment C.1085, CVB5, OLB5, C.M.B5, CXR5, CDR5, CIIb|35, CEB5, thereof, selected from CDH1 (E-cadherin, epithelial), CDH2 C.136, C.236, C3 B6, C4 B6, C.5 B6, C.6B6, C7f86, C.8f36, C.936, (N-cadherin, neural), CDH12 (cadherin 12, type 2, N-cad C. 1086, CVB6, CLB6, C.M.B6, CXR6, CDR6, CIIb?36, CEB6, herin 2), and CDH3 (P-cadherin, placental). In some C.1 f7, C.2B7, C3 B7, C4 B7, C.5 B7, C.6B7, C.7f87, C8B7, C.937, embodiments, a chimeric receptor polypeptide comprises a C.1087, CVB7, OLB7, C.M.B7, CXR7, CDR7, CIIb|37, CEB7, desmosoma cadherin, or any derivative, variant or fragment C.1? 8, O2B8, C3 B8, C4 B8, C.5 B8, C.6 B8, C7 B8, C8B8, C.938, thereof, selected from desmoglein (DSG1, DSG2. DSG3. C.1038, CVB8, CLB8, C.M.B8, CXP8, CDR8, CIIb?38, and DSG4) and desmocollin (DSC1, DSC2, DSC3). In some C.E.B8 receptor. A chimeric receptor polypeptide comprising embodiments, a chimeric receptor polypeptide comprises a an integrin subunit, or any derivative, variant or fragment protocadherin, or any derivative, variant or fragment thereof, can dimerize with an endogenous integrin subunit thereof, selected from PCDH1, PCDH10, PCDH11X, (e.g., wild-type integrin Subunit). PCDH11Y, PCDH12, PCDH15, PCDH17, PCDH18, 0143 A chimeric receptor polypeptide comprising an PCDH19, PCDH20, PCDH7, PCDH8, PCDH9, PCDHA1, integrin Subunit, or any derivative, variant or fragment PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHA2, thereof, can bind an antigen comprising any Suitable integrin PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, ligand, or any derivative, variant or fragment thereof. Non PCDHA8, PCDHA9, PCDHAC1, PCDHAC2, PCDHB1, limiting examples of ligands which can be bound by an PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, integrin receptor include adenovirus penton base protein, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHB2, beta-glucan, bone sialoprotein (BSP), Borrelia burgdorferi, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, Candida albicans, collagens (CN, e.g., CNI-IV), cytotactin/ PCDHB8, PCDHB9, PCDHGA1, PCDHGA10, PCD tenascin-C, decorsin, denatured collagen, disintegrins, HGA11, PCDHGA12, PCDHGA2, PCDHGA3, PCD E-cadherin, echovirus 1 receptor, epiligrin, Factor X, Fc HGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, epsilon RII (CD23), fibrin (Fb), fibrinogen (Fg), fibronectin PCDHGA9, PCDHGB1, PCDHGB2, PCDHGB3, PCD (Fn), heparin, HIV Tat protein, iC3b, intercellular adhesion HGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, molecule (e.g., ICAM-1,2,3,4,5), invasin, L1 cell adhesion PCDHGC4, PCDHGC5, FAT, FAT2, and FAT). In some molecule (L1-CAM), laminin, lipopolysaccharide (LPS), embodiments, a chimeric receptor polypeptide comprises an MAdCAM-1, matrix metalloproteinase-2 (MMPe), neutro unconventional cadherin selected from CDH4 (R-cadherin, phil inhibitory factor (NIF), osteopontin (OP or OPN), retinal), CDH5 (VE-cadherin, vascular endothelial), CDH6 plasminogen, prothrombin, sperm fertilin, thrombospondin (K-cadherin, kidney), CDH7 (cadherin 7, type 2). CDH8 (TSP), vascular cell adhesion molecule 1 (VCAM-1), Vit (cadherin 8, type 2), CDH9 (cadherin 9, type 2, T1-cad ronectin (VN or VTN), and von Willebrand factor (vWF). herin), CDH10 (cadherin 10, type 2, T2-cadherin), CDH11 0144. In some embodiments, a chimeric receptor poly (OB-cadherin, osteoblast), CDH13 (T-cadherin, H-cadherin, peptide comprises a cadherin molecule, or any derivative, heart), CDH15 (M-cadherin, myotubule), CDH16 (KSP variant or fragment thereof. Cadherin molecules, which can cadherin), CDH17 (LI cadherin, liver-intestine), CDH18 function as both ligands and receptors, refer to certain (cadherin 18, type 2), CDH19 (cadherin 19, type 2), CDH20 proteins involved in mediating cell adhesion. Cadherin (cadherin 20, type 2). CDH23 (cadherin 23, neurosensory molecules generally consist of five tandem repeated extra epithelium), CDH24, CDH26, CDH28, CELSR1, CELSR2, cellular domains, a single membrane-spanning segment and CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, a cytoplasmic region. E-cadherin, or CDH1, for example, LOC389118, PCLKC, RESDA1, and RET. consists of 5 repeats in the extracellular domain, one trans 0146 Achimeric receptor polypeptide comprising a cad membrane domain, and an intracellular domain. When herin, or any derivative, variant or fragment thereof, can E-cadherin is phosphorylated at a region of the intracellular bind an antigen comprising any suitable cadherin ligand, or domain, adaptor proteins such as beta-catenin and p120 any derivative, variant or fragment thereof. A cadherin catenin can bind to the receptor. In some embodiments, a ligand can comprise, for example, another cadherin receptor chimeric receptor polypeptide comprises at least an extra (e.g., a cadherin receptor of a cell). cellular region of a cadherin, or any derivative, variant or 0.147. In some embodiments, a chimeric receptor poly fragment thereof. In some embodiments, a chimeric receptor peptide comprises a catalytic receptor, or any derivative, polypeptide comprises at least a region spanning a mem variant or fragment thereof Examples of catalytic receptors brane of a cadherin, or any derivative, variant or fragment include, but are not limited to, receptor tyrosine kinases thereof. In some embodiments, a chimeric receptor poly (RTKs) and receptor threonine/serine kinases (RTSKs). peptide comprises at least an intracellular region (e.g., Catalytic receptors such as RTKs and RTSKs possess certain cytoplasmic domain) of a cadherin, or any derivative, vari enzymatic activities. RTKs, for example, can phosphorylate ant or fragment thereof. A chimeric receptor polypeptide Substrate proteins on tyrosine residues which can then act as comprising a cadherin, or any derivative, variant or fragment binding sites for adaptor proteins. RTKs generally comprise thereof, can recruit a binding partner. In some embodiments, an N-terminal extracellular ligand-binding domain, a single ligand binding to a chimeric receptor comprising a cadherin, transmembrane a helix, and a cytosolic C-terminal domain or any derivative, variant or fragment thereof, results in a with protein-tyrosine kinase activity. Some RTKs consist of conformational change, chemical modification, combination single polypeptides while Some are dimers consisting of two thereof, which recruits a binding partner to the receptor. pairs of polypeptide chains, for example the insulin receptor 0145 A chimeric receptor polypeptide can comprise a and some related receptors. The binding of ligands to the cadherin, or any derivative, variant or fragment thereof, extracellular domains of these receptors can activate the selected from a classical cadherin, a desmosoma cadherin, a cytosolic kinase domains, resulting in phosphorylation of US 2017/O 1983O8 A1 Jul. 13, 2017 both the receptors themselves and intracellular target pro 0150. A chimeric receptor polypeptide comprising a teins that propagate the signal initiated by ligand binding. In RTK, or any derivative, variant or fragment thereof, can bind Some RTKs, ligand binding induces receptor dimerization. an antigen comprising any suitable RTK ligand, or any Some ligands (e.g., growth factors such as PDGF and NGF) derivative, variant or fragment thereof. Non limiting are themselves dimers consisting of two identical polypep examples of RTK ligands include growth factors, cytokines, tide chains. These growth factors can directly induce and hormones. Growth factors include, for example, mem dimerization by simultaneously binding to two different bers of the epidermal growth factor family (e.g., epidermal receptor molecules. Other growth factors (e.g., such as EGF) growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-C. or TGF-C. are monomers but have two distinct receptor binding sites amphiregulin or AR, epiregulin or EPR, epigen, betacellulin that can crosslink receptors. Ligand-induced dimerization or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2. can result in autophosphorylation of the receptor, wherein neuregulin-3 or NRG3, and neuregulin-4 or NRG4), the the dimerized polypeptide chains cross-phosphorylate one fibroblast growth factor family (e.g., FGF1, FGF2, FGF3, another. Some receptors can multimerize. FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, 0148. In some embodiments, a chimeric receptor poly FGF12, FGF13, FGF14, FGF15/19, FGF16, FGF17, peptide comprises at least an extracellular region (e.g., FGF18, FGF20, FGF21, and FGF23), the vascular endothe ligand binding domain) of a catalytic receptor Such as a lial growth factor family (e.g., VEGF-A, VEGF-B, VEGF RTK, or any derivative, variant or fragment thereof. In some C, VEGF-D, and PIGF), and the platelet-derived growth embodiments, a chimeric receptor polypeptide comprises at factor family (e.g., PDGFA, PDGFB, PDGFC, and least a membrane spanning region of a catalytic receptor PDGFD). Hormones include, for example, members of the Such as a RTK, or any derivative, variant or fragment insulin/IGF/relaxin family (e.g., insulin, insulin-like growth thereof. In some embodiments, a chimeric receptor poly factors, relaxin family peptides including relaxin1, relaxin2, peptide comprises at least an intracellular region (e.g., relaxin3, Leydig cell-specific insulin-like peptide (gene cytosolic domain) of a catalytic receptor Such as a RTK, or INSL3), early placenta insulin-like peptide (ELIP) (gene any derivative, variant or fragment thereof. A chimeric INSL4), insulin-like peptide 5 (gene INSL5), and insulin receptor polypeptide comprising an RTK, or any derivative, like peptide 6). variant or fragment thereof, can recruit a binding partner. In 0151. In some embodiments, a chimeric receptor poly Some embodiments, ligand binding to a chimeric receptor peptide comprises at least an extracellular region (e.g., comprising an RTK, or any derivative, variant or fragment ligand binding domain) of a catalytic receptor Such as an thereof, results in a conformational change, chemical modi RTSK, or any derivative, variant or fragment thereof. In fication, or combination thereof, which recruits a binding Some embodiments, a chimeric receptor polypeptide com partner to the receptor. prises at least a membrane spanning region of a catalytic 0149. In some embodiments, the chimeric receptor poly receptor such as an RTSK, or any derivative, variant or peptide comprises a class I RTK (e.g., the epidermal growth fragment thereof. In some embodiments, a chimeric receptor factor (EGF) receptor family including EGFR: the ErbB polypeptide comprises at least an intracellular region (e.g., family including ErbB-2, ErbB-3, and ErbB-4), a class II cytosolic domain) of a catalytic receptor Such as an RTSK, RTK (e.g., the insulin receptor family including INSR, or any derivative, variant or fragment thereof. A chimeric IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived receptor polypeptide comprising an RTSK, or any deriva growth factor (PDGF) receptor family including PDGFR-O. tive, variant or fragment thereof, can recruit a binding PDGFR-B, CSF-1R, KIT/SCFR, and FLK2/FLT3), a class partner. In some embodiments, ligand binding to a chimeric IV RTK (e.g., the fibroblast growth factor (FGF) receptor receptor comprising an RTSK, or any derivative, variant or family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), fragment thereof, results in a conformational change, chemi a class V RTK (e.g., the vascular endothelial growth factor cal modification, or combination thereof, which recruits a (VEGF) receptor family including VEGFR1, VEGFR2, and binding partner to the receptor. VEGFR3), a class VI RTK (e.g., the hepatocyte growth 0152. A chimeric receptor polypeptide comprising an factor (HGF) receptor family including hepatocyte growth RTSK, or any derivative, variant or fragment thereof, can factor receptor (HGFR/MET) and RON), a class VII RTK phosphorylate a Substrate at serine and/or threonine resi (e.g., the tropomyosin receptor kinase (Trk) receptor family dues, and may select specific residues based on a consensus including TRKA, TRKB, and TRKC), a class VIII RTK sequence. A chimeric receptor polypeptide can comprise a (e.g., the ephrin (Eph) receptor family including EPHA1, type I RTSK, type II RTSK, or any derivative, variant or EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, fragment thereof. In some embodiments, a chimeric receptor EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and polypeptide comprising a type I receptor serine/threonine EPHB6), a class IX RTK (e.g., AXL receptor family such as kinase is inactive unless complexed with a type II receptor. AXL, MER, and TRYO3), a class X RTK (e.g., LTK In some embodiments, a chimeric receptor polypeptide receptor family such as LTK and ALK), a class XI RTK comprising a type II receptor serine/threonine comprises a (e.g., TIE receptor family such as TIE and TEK), a class XII constitutively active kinase domain that can phosphorylate RTK (e.g., ROR receptor family ROR1 and ROR2), a class and activate a type I receptor when complexed with the type XIII RTK (e.g., the discoidin domain receptor (DDR) family I receptor. A type II receptor serine/threonine kinase can such as DDR1 and DDR2), a class XIV RTK (e.g., RET phosphorylate the kinase domain of the type I partner, receptor family such as RET), a class XV RTK (e.g., KLG causing displacement of protein partners. Displacement of receptor family including PTK7), a class XVI RTK (e.g., protein partners can allow binding and phosphorylation of RYK receptor family including Ryk), a class XVII RTK other proteins, for example certain members of the SMAD (e.g., MuSK receptor family such as MuSK), or any deriva family. A chimeric receptor polypeptide can comprise a type tive, variant or fragment thereof. I receptor, or any derivative, variant or fragment thereof, US 2017/O 1983O8 A1 Jul. 13, 2017 20 selected from the group consisting of ALK1 (ACVRL1), tive, variant or fragment thereof. In some embodiments, the ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), chimeric receptor polypeptide comprises an interferon ALK5 (TGFBR1), ALK6 (BMPR1B), and ALK7 receptor (e.g., IFNAR1, IFNAR2, and IFNGR), an interleu (ACVR1C). A chimeric receptor polypeptide can comprise kin receptor (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), a a type II receptor, or any derivative, variant or fragment tissue factor receptor (also called platelet tissue factor), or thereof, selected from the group consisting of TGFBR2, any derivative, variant or fragment thereof. BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR). In 0.155. A chimeric receptor polypeptide comprising a Some embodiments, a chimeric receptor polypeptide com cytokine receptor can bind an antigen comprising any Suit prises a TGF-B receptor, or any derivative, variant or frag able cytokine receptor ligand, or any derivative, variant or ment thereof. fragment thereof. Non-limiting examples of cytokine recep 0153. In some embodiments, a chimeric receptor poly tor ligands include interleukins (e.g., IL-2, IL-3, IL-4, IL-5, peptide comprises a receptor which stimulates non-cova IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-20, lently associated intracellular kinases, such as a Src kinase IL-21, IL-22, IL-23, IL-27, IL-28, and IL-31), interferons (e.g., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk) or (e.g., IFN-O, IFN-B, IFN-Y), colony stimulating factors (e.g., a JAK kinase (e.g., JAK1, JAK2, JAK3, and TYK2) rather erythropoietin, macrophage colony-stimulating factor, than possessing intrinsic enzymatic activity, or any deriva granulocyte macrophage colony-stimulating factors or GM tive, variant or fragment thereof. These include the cytokine CSFs, and granulocyte colony-stimulating factors or receptor Superfamily such as receptors for cytokines and G-CSFs), and hormones (e.g., prolactin and leptin). polypeptide hormones. Cytokine receptors generally contain 0156. In some embodiments, a chimeric receptor poly an N-terminal extracellular ligand-binding domain, trans peptide comprises a death receptor, a receptor containing a membrane a helices, and a C-terminal cytosolic domain. The death domain, or any derivative, variant or fragment thereof. cytosolic domains of cytokine receptors are generally devoid Death receptors are often involved in regulating apoptosis of any known catalytic activity. Cytokine receptors instead and inflammation. Death receptors include members of the can function in association with non-receptor kinases (e.g., TNF receptor family such as TNFR1, Fas receptor, DR4 tyrosine kinases or threonine/serine kinases), which can be (also known as TRAIL receptor 1 or TRAILR1) and DR5 activated as a result of ligand binding to the receptor. In (also known as TRAIL receptor 2 or TRAILR2). In some Some embodiments, a chimeric receptor polypeptide com embodiments, a chimeric receptor polypeptide comprises at prises at least an extracellular region (e.g., ligand binding least an extracellular region (e.g., ligand binding domain) of domain) of a catalytic receptor that non-covalently associ a death receptor, or any derivative, variant or fragment ates with an intracellular kinase (e.g., a cytokine receptor), thereof. In some embodiments, a chimeric receptor poly or any derivative, variant or fragment thereof. In some peptide comprises at least a membrane spanning region of a embodiments, a chimeric receptor polypeptide comprises at death receptor, or any derivative, variant or fragment least a membrane spanning region of a catalytic receptor that thereof. In some embodiments, a chimeric receptor poly non-covalently associates with an intracellular kinase (e.g., peptide comprises at least an intracellular region (e.g., a cytokine receptor), or any derivative, variant or fragment cytosolic) domain of a death receptor, or any derivative, thereof. In some embodiments, a chimeric receptor poly variant or fragment thereof. A chimeric receptor polypeptide peptide comprises at least an intracellular region (e.g., comprising a death receptor, or any derivative, variant or cytosolic domain) of a catalytic receptor that non-covalently fragment thereof, can undergo receptor oligomerization in associates with an intracellular kinase (e.g., a cytokine response to ligand binding, which in turn can result in the receptor), or any derivative, variant or fragment thereof. A recruitment of specialized adaptor proteins and activation of chimeric receptor polypeptide comprising a catalytic recep signaling cascades, such as caspase cascades. In some tor that non-covalently associates with an intracellular embodiments, a chimeric receptor polypeptide comprises a kinase, or any derivative, variant or fragment thereof, can death receptor, or any derivative, variant or fragment recruit a binding partner. In some embodiments, ligand thereof, results in a conformational change, chemical modi binding to a chimeric receptor comprising a catalytic recep fication, or combination thereof, which recruits a binding tor that non-covalently associates with an intracellular partner to the receptor. kinase, or any derivative, variant or fragment thereof, results 0157. A chimeric receptor polypeptide comprising a in a conformational change, chemical modification, or com death receptor can bind an antigen comprising any Suitable bination thereof, which recruits a binding partner to the ligand of a death receptor, or any derivative, variant or receptor. fragment thereof. Non-limiting examples of ligands bound 0154) In some embodiments, a chimeric receptor poly by death receptors include TNFO, Fas ligand, and TNF peptide comprises a cytokine receptor, for example a type I related apoptosis-inducing ligand (TRAIL). cytokine receptor or a type II cytokine receptor, or any 0158. In some embodiments, a chimeric receptor poly derivative, variant or fragment thereof. In some embodi peptide comprises an immune receptor, or any derivative, ments, the chimeric receptor polypeptide comprises an inter variant or fragment thereof. Immune receptors include mem leukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R. bers of the immunoglobulin superfamily (IgSF) which share IL-7R, IL-9R, IL-11 R, IL-12R, IL-13R, IL-15R, IL-21R, structural features with immunoglobulins, e.g., a domain IL-23R, IL-27R, and IL-31R), a colony stimulating factor known as an immunoglobulin domain or fold. IgSF mem receptor (e.g., erythropoietin receptor, CSF-1R, CSF-2R, bers include, but are not limited to, cell Surface antigen GM-CSFR, and G-CSFR), a hormone receptor/neuropeptide receptors, co-receptors and costimulatory molecules of the receptor (e.g., growth hormone receptor, prolactin receptor, immune system, and molecules involved in antigen presen and leptin receptor), or any derivative, variant or fragment tation to lymphocytes. In some embodiments, a chimeric thereof. In some embodiments, the chimeric receptor poly receptor polypeptide comprises at least an extracellular peptide comprises a type II cytokine receptor, or any deriva region (e.g., ligand binding domain) of an immune receptor, US 2017/O 1983O8 A1 Jul. 13, 2017

or any derivative, variant or fragment thereof. In some interactions between the cell signals that bind to the recom embodiments, a chimeric receptor polypeptide comprises at binant chimeric receptor polypeptides involve a cell-cell least a region spanning a membrane of an immune receptor, interaction, cell-soluble chemical interaction, and cell-ma or any derivative, variant or fragment thereof. In some trix or microenvironment interaction. embodiments, a chimeric receptor polypeptide comprises at 0.161. A gene modulating polypeptide (GMP) of a chi least an intracellular region (e.g., cytoplasmic domain) of an meric receptor polypeptide can comprise an actuator moiety immune receptor, or any derivative, variant or fragment linked to a cleavage recognition site. The actuator moiety thereof. A chimeric receptor polypeptide comprising an can comprise a nuclease (e.g., DNA nuclease and/or RNA immune receptor, or any derivative, variant or fragment nuclease), modified nuclease (e.g., DNA nuclease and/or thereof, can recruit a binding partner. In some embodiments, RNA nuclease) that is nuclease-deficient or has reduced ligand binding to a chimeric receptor comprising an immune nuclease activity compared to a wild-type nuclease, a deriva receptor, or any derivative, variant or fragment thereof, tive thereof, a variant thereof, or a fragment thereof. The results in a conformational change, chemical modification, actuator moiety can regulate expression or activity of a gene or combination thereof, which recruits a binding partner to and/or edit the sequence of a nucleic acid (e.g., a gene and/or the receptor. gene product). In some embodiments, the actuator moiety 0159. In some embodiments, a chimeric receptor poly comprises a DNA nuclease Such as an engineered (e.g., peptide comprises a cell Surface antigen receptor Such as a programmable or targetable) DNA nuclease to induce T cell receptor (TCR), a B cell receptor (BCR), or any genome editing of a target DNA sequence. In some embodi derivative, variant or fragment thereof. T cell receptors ments, the actuator moiety comprises a RNA nuclease Such generally comprise two chains, either the TCR-alpha and as an engineered (e.g., programmable or targetable) RNA -beta chains or the TCR-delta and -gamma chains. A chi nuclease to induce editing of a target RNA sequence. In meric receptor polypeptide comprising a TCR, or any Some embodiments, the actuator moiety has reduced or derivative, variant or fragment thereof, can bind a major minimal nuclease activity. An actuator moiety having histocompatibility complex (MHC) protein. B cell receptors reduced or minimal nuclease activity can regulate expres generally comprises a membrane bound immunoglobulin sion and/or activity of a gene by physical obstruction of a and a signal transduction moiety. A chimeric receptor com target polynucleotide or recruitment of additional factors prising a BCR, or any derivative, variant or fragment effective to Suppress or enhance expression of the target thereof, can bind a cognate BCR antigen. In some embodi polynucleotide. In some embodiments, the actuator moiety ments, a chimeric receptor polypeptide comprises at least an comprises a nuclease-null DNA binding protein derived immunoreceptor tyrosine-based activation motif (ITAM) from a DNA nuclease that can induce transcriptional acti found in the cytoplasmic domain of certain immune recep Vation or repression of a target DNA sequence. In some tors. In some embodiments, a chimeric receptor polypeptide embodiments, the actuator moiety comprises a nuclease-null comprises at least an immunoreceptor tyrosine-based inhi RNA binding protein derived from a RNA nuclease that can bition motif (ITIM) found in the cytoplasmic domain of induce transcriptional activation or repression of a target certain immune receptors. Chimeric receptor polypeptides RNA sequence. In some embodiments, the actuator moiety comprising ITAM and/or ITIM domains can be phosphory is a nucleic acid-guided actuator moiety. In some embodi lated following ligand binding to an antigen interacting ments, the actuator moiety is a DNA-guided actuator moiety. domain. The phosphorylated regions can serve as docking In some embodiments, the actuator moiety is an RNA sites for other proteins involved in immune cell signaling. guided actuator moiety. An actuator moiety can regulate 0160 The antigen interacting domain of a chimeric expression or activity of a gene and/or edit a nucleic acid receptor polypeptide can bind a membrane bound antigen, sequence, whether exogenous or endogenous. for example an antigen bound to the extracellular Surface of 0162 Any suitable nuclease can be used in an actuator a cell (e.g., a target cell). In some embodiments, the antigen moiety. Suitable nucleases include, but are not limited to, interacting domain binds a non-membrane bound antigen, CRISPR-associated (Cas) proteins or Cas nucleases includ for example an extracellular antigen that is secreted by a cell ing type I CRISPR-associated (Cas) polypeptides, type II (e.g., a target cell) or an antigen located in the cytoplasm of CRISPR-associated (Cas) polypeptides, type III CRISPR a cell. Antigens (e.g., membrane bound and non-membrane associated (Cas) polypeptides, type IV CRISPR-associated bound) can be associated with a disease Such as a viral, (Cas) polypeptides, type V CRISPR-associated (Cas) poly bacterial, and/or parasitic infection; inflammatory and/or peptides, and type VI CRISPR-associated (Cas) polypep autoimmune disease; or neoplasm such as a cancer and/or tides; Zinc finger nucleases (ZFN); transcription activator tumor. Cancer antigens, for example, are proteins produced like effector nucleases (TALEN); meganucleases; RNA by tumor cells that can elicit an immune response, particu binding proteins (RBP); CRISPR-associated RNA binding larly a T-cell mediated immune response. The selection of proteins; recombinases; flippases; transposases; Argonaute the antigen binding portions of a chimeric receptor poly (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), peptide can depend on the particular type of cancer antigen archaeal Argonaute (aAgo), and eukaryotic Argonaute to be targeted. In some embodiments, the tumor antigen (eAgo)); any derivative thereof, any variant thereof, and any comprises one or more antigenic cancer epitopes associated fragment thereof. with a malignant tumor. Malignant tumors can express a 0163 The regulation of genes can be of any gene of number of proteins that can serve as target antigens for an interest. It is contemplated that genetic homologues of a immune attack. The antigen interaction domains can bind to gene described herein are covered. For example, a gene can cell Surface signals, extracellular matrix (ECM), paracrine exhibit a certain identity and/or homology to genes disclosed signals, juxtacrine signals, endocrine signals, autocrine sig herein. Therefore, it is contemplated that a gene that exhibits nals, signals that can trigger or control genetic programs in or exhibits about 50%, 55%, 60%, 65%,70%, 75%, 80%, cells, or any combination thereof. In some embodiments, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, US 2017/O 1983O8 A1 Jul. 13, 2017 22

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% target polynucleotide and prevent transcription by physical homology (at the nucleic acid or protein level) can be obstruction or edit a nucleic acid sequence to yield non modified. It is also contemplated that a gene that exhibits or functional gene products. exhibits about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 0167. In some embodiments, the actuator moiety com 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, prises a Cas protein that forms a complex with a guide 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid, such as a guide RNA. In some embodiments, identity (at the nucleic acid or protein level) can be modified. the actuator moiety comprises a Cas protein that forms a 0164. In some embodiments, the actuator moiety com complex with a single guide nucleic acid, Such as a single prises a CRISPR-associated (Cas) protein or a Cas nuclease guide RNA (sgRNA). In some embodiments, the actuator which functions in a non-naturally occurring CRISPR (Clus moiety comprises a RNA-binding protein (RBP) optionally tered Regularly Interspaced Short Palindromic Repeats)/Cas complexed with a guide nucleic acid. Such as a guide RNA (CRISPR-associated) system. In bacteria, this system can (e.g., SgRNA), which is able to form a complex with a Cas provide adaptive immunity against foreign DNA (Barran protein. FIG. 3A illustrates schematically a system compris gou, R., et al., “CRISPR provides acquired resistance against ing a chimeric receptor polypeptide in which the actuator viruses in prokaryotes.” Science (2007) 315: 1709-1712; moiety comprises an RNA-binding protein 300a optionally Makarova, K. S., et al., “Evolution and classification of the complexed with a guide nucleic acid (e.g., sgRNA). Upon CRISPR-Cas systems.” Nat Rev. Microbiol (2011) 9:467 release from the RNA-binding protein (RBP), for example 477; Garneau, J. E., et al., “The CRISPR/Cas bacterial by dissociation of the guide nucleic acid from the RBP or immune system cleaves bacteriophage and plasmid DNA.” cleavage of the cleavage recognition site 300c, the guide Nature (2010) 468:67-71 : Sapranauskas, R., et al., “The nucleic acid can form a complex with a Cas protein 300b Streptococcus thermophilus CRISPR/Cas system provides which is operable to regulate gene expression and/or activity immunity in Escherichia coli,' Nucleic Acids Res (2011) or to edit a nucleic acid sequence. In some embodiments, the 39:9275-9282). actuator moiety comprises a nuclease-null DNA binding protein derived from a DNA nuclease that can induce 0.165. In a wide variety of organisms including diverse transcriptional activation or repression of a target DNA mammals, animals, plants, and yeast, a CRISPR/Cas system sequence. In some embodiments, the actuator moiety com (e.g., modified and/or unmodified) can be utilized as a prises a nuclease-null RNA binding protein derived from a genome engineering tool. A CRISPR/Cas system can com RNA nuclease that can induce transcriptional activation or prise a guide nucleic acid such as a guide RNA (gRNA) repression of a target RNA sequence. For example, an complexed with a Cas protein for targeted regulation of gene actuator moiety can comprise a Cas protein which lacks expression and/or activity or nucleic acid editing. An RNA cleavage activity. guided Cas protein (e.g., a Cas nuclease such as a Cas9 0168 Any suitable CRISPR/Cas system can be used. A nuclease) can specifically bind a target polynucleotide (e.g., CRISPR/Cas system can be referred to using a variety of DNA) in a sequence-dependent manner. The Cas protein, if naming systems. Exemplary naming systems are provided in possessing nuclease activity, can cleave the DNA (Gasiunas, Makarova, K. S. et al., “An updated evolutionary classifica G. et al., “Cas9-crRNA ribonucleoprotein complex mediates tion of CRISPR-Cas systems.” Nat Rev. Microbiol (2015) specific DNA cleavage for adaptive immunity in bacteria.” 13:722-736 and Shmakov, S. et al., “Discovery and Func Proc Natl Acad Sci USA (2012) 109: E2579-E2 86; Jinek, tional Characterization of Diverse Class 2 CRISPR-Cas M., et al., “A programmable dual-RNA-guided DNA endo Systems.” Mol Cell (2015) 60:1-13. A CRISPR/Cas system nuclease in adaptive bacterial immunity.” Science (2012) can be a type I, a type II, a type III, a type IV, a type V, a 337:816-821; Sternberg, S. H., et al., “DNA interrogation by type VI system, or any other suitable CRISPR/Cas system. the CRISPR RNA-guided endonuclease Cas9,” Nature ACRISPR/Cas system as used herein can be a Class 1, Class (2014) 507:62: Deltcheva, E., et al., “CRISPR RNA matu 2, or any other suitably classified CRISPR/Cas system. Class ration by trans-encoded small RNA and host factor RNase 1 or Class 2 determination can be based upon the genes III,” Nature (2011) 471:602-607), and has been widely used encoding the effector module. Class 1 systems generally for programmable genome editing in a variety of organisms have a multi-subunit crRNA-effector complex, whereas and model systems (Cong, L., et al., “Multiplex genome Class 2 systems generally have a single protein, Such as engineering using CRISPR Cas systems.” Science (2013) Cas9, Cpf1, C2c1, C2c2, C2c3 or a crRNA-effector com 339:819-823; Jiang, W., et al., “RNA-guided editing of plex. A Class 1 CRISPR/Cas system can use a complex of bacterial genomes using CRISPR-Cas systems.” Nat. Bio multiple Cas proteins to effect regulation. A Class 1 technol. (2013) 31 : 233-239; Sander, J. D. & Joung, J. K. CRISPR/Cas system can comprise, for example, type I (e.g., “CRISPR-Cas systems for editing, regulating and targeting I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, genomes.” Nature Biotechnol. (2014) 32:347-355). IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR/Cas 0166 In some cases, the Cas protein is mutated and/or type. A Class 2 CRISPR/Cas system can use a single large modified to yield a nuclease deficient protein or a protein Cas protein to effect regulation. A Class 2 CRISPR/Cas with decreased nuclease activity relative to a wild-type Cas systems can comprise, for example, type II (e.g., II, IIA, IIB) protein. A nuclease deficient protein can retain the ability to and type V CRISPR/Cas type. CRISPR systems can be bind DNA, but may lack or have reduced nucleic acid complementary to each other, and/or can lend functional cleavage activity. An actuator moiety comprising a Cas units in trans to facilitate CRISPR locus targeting. FIG. 15 nuclease (e.g., retaining wild-type nuclease activity, having shows an illustration adapted from FIG. 2 of Makarova, K. reduced nuclease activity, and/or lacking nuclease acitivity) S. et al., “An updated evolutionary classification of CRISPR can function in a CRISPR/Cas system to regulate the level Cas systems.” Nat Rev Microbiol (2015) 13:722-736 pro and/or activity of a target gene or protein (e.g., decrease, viding architectures of the genomic loci for Subtypes of increase, or elimination). The Cas protein can bind to a CRISPR-Cas systems. US 2017/O 1983O8 A1 Jul. 13, 2017

0169. An actuator moiety comprising a Cas protein can ticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, be a Class 1 or a Class 2 Cas protein. A Cas protein can be Streptococcus mutans, Listeria innocua, Staphylococcus a type I, type II, type III, type IV, type V Cas protein, or type pseudintermedius, Acidaminococcus intestine, Olsenella uli, VI Cas protein. A Cas protein can comprise one or more Oenococcus kiltaharae, Bifidobacterium bifidum, Lactoba domains. Non-limiting examples of domains include, guide cillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, nucleic acid recognition and/or binding domain, nuclease Mycoplasma mobile, Mycoplasma gallisepticum, Myco domains (e.g., DNase or RNase domains, RuvC, HNH), plasma ovipneumoniae, Mycoplasma canis, Mycoplasma DNA binding domain, RNA binding domain, helicase synoviae, Eubacterium rectale, Streptococcus thermophilus, domains, protein-protein interaction domains, and dimeriza Eubacterium dolichum, Lactobacillus coryniformis Subsp. tion domains. A guide nucleic acid recognition and/or bind Torquens, Ilvobacter polytropus, Ruminococcus albus, ing domain can interact with a guide nucleic acid. A nuclease Akkermansia muciniphila, Acidothermus cellulolyticus, Bifi domain can comprise catalytic activity for nucleic acid dobacterium longum, Bifidobacterium dentium, Corynebac cleavage. A nuclease domain can lack catalytic activity to terium diphtheria, Elusimicrobium minutum, Nitratifractor prevent nucleic acid cleavage. A Cas protein can be a salsuginis, Sphaerochaeta globus, Fibrobacter succino chimeric Cas protein that is fused to other proteins or genes Subsp. Succinogenes, Bacteroides fragilis, Capnocy polypeptides. A Cas protein can be a chimera of various Cas tophaga ochracea, Rhodopseudomonas palustris, PrevOtella proteins, for example, comprising domains from different micans, PrevOtella ruminicola, Flavobacterium columnare, Cas proteins. Aminomonas paucivorans, Rhodospirillum rubrum, Candi 0170 Non-limiting examples of Cas proteins include datus Puniceispirillum marinum, Verminephrobacter eise c2c1, C2c2, c2c3, Cas1, Cas 1B, Cas2, Cas3, Cas4, Cas5, niae, Ralstonia syzygii, Dinoroseobacter Shibae, Azospiril Cas5e (CasD), Casó, Casóe, Caséf, Cas7, Cas8a, Cas8a1, lum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or CSX12), Cas10, succinogenes, Campylobacter jejuni Subsp. Jejuni, Helico Cas 10d, Cas 1C, Cas 1Cd, Cash, CasG, Cash, Cpf1, Csy 1, bacter mustelae, Bacillus cereus, Acidovorax ebreus, Csy2, Csy3, Cse 1 (CasA), Cse2 (Casb), Cse3 (Cash), Cse4 Clostridium perfingens, Parvibaculum lavamentivorans, (CasO), Csc1, Csc2, Csa5, Csn2, Csm2, CSm3, Csm4. Roseburia intestinalis, Neisseria meningitidis, Pasteurella Csm5, Csm6, Cmr1, Cmr3, Cmra, CmriS, Cmr6, Csb1, multocida Subsp. Multocida, Sutterella Wadsworthensis, Csb2, Csb3, CSX17, CSX14, CSx1O, CSX16, Csax, CSX3, proteobacterium, Legionella pneumophila, Parasutterella Csx1, CSX15, Csf1, CSf2, Csf3, Csf4, and Cu1966, and excrementihominis, Wolinella succinogenes, and Francisella homologs or modified versions thereof. novicida. 0171 A Cas protein can be from any suitable organism. 0173 A Cas protein as used herein can be a wildtype or Non-limiting examples include Streptococcus pyogenes, a modified form of a Cas protein. A Cas protein can be an Streptococcus thermophilus, Streptococcus sp., Staphyllo active variant, inactive variant, or fragment of a wild type or coccus aureus, Nocardiopsis dassonvillei, Streptomyces modified Cas protein. A Cas protein can comprise an amino pristinae spiralis, Streptomyces viridochromo genes, Strep acid change such as a deletion, insertion, Substitution, Vari tomyces viridochromogenes, Streptosporangium roseum, ant, mutation, fusion, chimera, or any combination thereof Streptosporangium roseum, AlicyclobacHlus acidocal relative to a wild-type version of the Cas protein. A Cas darius, Bacillus pseudomycoides, Bacillus selenitireducens, protein can be a polypeptide with at least about 5%, 10%, Exiguobacterium Sibiricum, Lactobacillus delbruecki, Lac 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, tobacillus salivarius, Microscilla marina, Burkholderiales 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence bacterium, PolarOmonas naphthalenivorans, Polaromonas identity or sequence similarity to a wild type exemplary Cas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis protein. A Cas protein can be a polypeptide with at most aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, Acetohalobium arabaticum, Ammonifex degensii, Caldice 90%, 100% sequence identity and/or sequence similarity to lulosiruptor becscii, Candidatus Desulforudis, Clostridium a wild type exemplary Cas protein. Variants or fragments botulinum, Clostridium difficile, Finegoldia magna, Natran can comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, aerobius thermophilus, Pelotomaculum thermopropioni 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, cum, Acidithiobacillus caldus, Acidithiobacillus ferrooxi 97%, 98%, 99%, or 100% sequence identity or sequence dans, Allochromatium vinosum, Marinobacter sp., similarity to a wild type or modified Cas protein or a portion Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudo thereof. Variants or fragments can be targeted to a nucleic alteromonas haloplanktis, Ktedonobacter racemifer, Metha acid locus in complex with a guide nucleic acid while nohalobium evestigatum, Anabaena variabilis, Nodularia lacking nucleic acid cleavage activity. spunigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chtho 0.174 A Cas protein can comprise one or more nuclease noplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho domains, such as DNase domains. For example, a Cas9 africanus, Acaryochloris marina, Leptotrichia Shahii, and protein can comprise a RuvC-like nuclease domain and/or Francisella novicida. In some aspects, the organism is an HNH-like nuclease domain. The RuvC and HNH Streptococcus pyogenes (S. pyogenes). In some aspects, the domains can each cut a different strand of double-stranded organism is Staphylococcus aureus (S. aureus). In some DNA to make a double-stranded break in the DNA. A Cas aspects, the organism is Streptococcus thermophilus (S. protein can comprise only one nuclease domain (e.g., Cpfl thermophilus). comprises RuvC domain but lacks HNH domain). 0172 A Cas protein can be derived from a variety of 0.175. A Cas protein can comprise an amino acid bacterial species including, but not limited to, Veillonella sequence having at least about 5%, 10%, 20%, 30%, 40%, atypical, Fusobacterium nucleatum, Filifactor alocis, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, Solobacterium moorei, Coprococcus catus, Treponema den 96%, 97%, 98%, 99%, or 100% sequence identity or US 2017/O 1983O8 A1 Jul. 13, 2017 24 sequence similarity to a nuclease domain (e.g., RuvC the engineered chimeric receptor polypeptide. In some domain, HNH domain) of a wild-type Cas protein. cases, such cells contain one or more different sgRNAS that 0176 A Cas protein can be modified to optimize regula target the same nucleic acid. In other cases, the sgRNAS tion of gene expression. A Cas protein can be modified to target different nucleic acids in the cell. The nucleic acids increase or decrease nucleic acid binding affinity, nucleic targeted by the guide RNA can be any that are expressed in acid binding specificity, and/or enzymatic activity. Cas pro a cell Such as an immune cell. The nucleic acids targeted teins can also be modified to change any other activity or may be a gene involved in immune cell regulation. In some property of the protein, Such as Stability. For example, one embodiments, the nucleic acid is associated with cancer. The or more nuclease domains of the Cas protein can be modi nucleic acid associated with cancer can be a cell cycle gene, fied, deleted, or inactivated, or a Cas protein can be trun cell response gene, apoptosis gene, or phagocytosis gene. cated to remove domains that are not essential for the The recombinant guide RNA can be recognized by a function of the protein or to optimize (e.g., enhance or CRISPR protein, a nuclease-null CRISPR protein, variants reduce) the activity of the Cas protein for regulating gene thereof, or derivatives thereof. expression. 0183 Enzymatically inactive can refer to a polypeptide 0177. A Cas protein can be a fusion protein. For example, that can bind to a nucleic acid sequence in a polynucleotide a Cas protein can be fused to a cleavage domain, an in a sequence-specific manner, but may not cleave a target epigenetic modification domain, a transcriptional activation polynucleotide. An enzymatically inactive site-directed domain, or a transcriptional repressor domain. A Cas protein polypeptide can comprise an enzymatically inactive domain can also be fused to a heterologous polypeptide providing (e.g. nuclease domain). Enzymatically inactive can refer to increased or decreased stability. The fused domain or het no activity. Enzymatically inactive can refer to Substantially erologous polypeptide can be located at the N-terminus, the no activity. Enzymatically inactive can refer to essentially no C-terminus, or internally within the Cas protein. activity. Enzymatically inactive can refer to an activity less 0.178 A Cas protein can be provided in any form. For than 1%, less than 2%, less than 3%, less than 4%, less than example, a Cas protein can be provided in the form of a 5%, less than 6%, less than 7%, less than 8%, less than 9%, protein, Such as a Cas protein alone or complexed with a or less than 10% activity compared to a wild-type exemplary guide nucleic acid. A Cas protein can be provided in the form activity (e.g., nucleic acid cleaving activity, wild-type Cas9 of a nucleic acid encoding the Cas protein, Such as an RNA activity). (e.g., messenger RNA (mRNA)) or DNA. The nucleic acid 0.184 One or a plurality of the nuclease domains (e.g., encoding the Cas protein can be codon optimized for effi RuvC, HNH) of a Cas protein can be deleted or mutated so cient translation into protein in a particular cell or organism. that they are no longer functional or comprise reduced 0179 Nucleic acids encoding Cas proteins can be stably nuclease activity. For example, in a Cas protein comprising integrated in the genome of the cell. Nucleic acids encoding at least two nuclease domains (e.g., Cas9), if one of the Cas proteins can be operably linked to a promoter active in nuclease domains is deleted or mutated, the resulting Cas the cell. Nucleic acids encoding Cas proteins can be oper protein, known as a nickase, can generate a single-strand ably linked to a promoter in an expression construct. Expres break at a CRISPR RNA (crRNA) recognition sequence sion constructs can include any nucleic acid constructs within a double-stranded DNA but not a double-strand capable of directing expression of a gene or other nucleic break. Such a nickase can cleave the complementary strand acid sequence of interest (e.g., a Cas gene) and which can or the non-complementary Strand, but may not cleave both. transfer Such a nucleic acid sequence of interest to a target If all of the nuclease domains of a Cas protein (e.g., both cell. RuvC and HNH nuclease domains in a Cas9 protein; RuvC 0180. In some embodiments, a Cas protein is a dead Cas nuclease domain in a Cpfl protein) are deleted or mutated, protein. A dead Cas protein can be a protein that lacks the resulting Cas protein can have a reduced or no ability to nucleic acid cleavage activity. cleave both strands of a double-stranded DNA. An example 0181 A Cas protein can comprise a modified form of a of a mutation that can convert a Cas9 protein into a nickase wild type Cas protein. The modified form of the wild type is a D10A (aspartate to alanine at position 10 of Cas9) Cas protein can comprise an amino acid change (e.g., mutation in the RuvC domain of Cas9 from S. pyogenes. deletion, insertion, or Substitution) that reduces the nucleic H939A (histidine to alanine at amino acid position 839) or acid-cleaving activity of the Cas protein. For example, the H840A (histidine to alanine at amino acid position 840) in modified form of the Cas protein can have less than 90%, the HNH domain of Cas9 from S. pyogenes can convert the less than 80%, less than 70%, less than 60%, less than 50%, Cas9 into a nickase. An example of a mutation that can less than 40%, less than 30%, less than 20%, less than 10%, convert a Cas9 protein into a dead Cas9 is a D10A (aspartate less than 5%, or less than 1% of the nucleic acid-cleaving to alanine at position 10 of Cas9) mutation in the RuvC activity of the wild-type Cas protein (e.g., Cas9 from S. domain and H939A (histidine to alanine at amino acid pyogenes). The modified form of Cas protein can have no position 839) or H840A (histidine to alanine at amino acid Substantial nucleic acid-cleaving activity. When a Cas pro position 840) in the HNH domain of Cas9 from S. pyogenes. tein is a modified form that has no Substantial nucleic 0185. A dead Cas protein can comprise one or more acid-cleaving activity, it can be referred to as enzymatically mutations relative to a wild-type version of the protein. The inactive and/or “dead” (abbreviated by “d'). A dead Cas mutation can result in less than 90%, less than 80%, less than protein (e.g., dCas, dCas9) can bind to a target polynucle 70%, less than 60%, less than 50%, less than 40%, less than otide but may not cleave the target polynucleotide. In some 30%, less than 20%, less than 10%, less than 5%, or less than aspects, a dead Cas protein is a dead Cas9 protein. 1% of the nucleic acid-cleaving activity in one or more of 0182. A dCas9 polypeptide can associate with a single the plurality of nucleic acid-cleaving domains of the wild guide RNA (sgRNA) to activate or repress transcription of type Cas protein. The mutation can result in one or more of target DNA. sgRNAs can be introduced into cells expressing the plurality of nucleic acid-cleaving domains retaining the US 2017/O 1983O8 A1 Jul. 13, 2017 ability to cleave the complementary Strand of the target larity to a wild type exemplary Cas9 polypeptide (e.g., from nucleic acid but reducing its ability to cleave the non S. pyogenes). Cas9 can refer to the wildtype or a modified complementary strand of the target nucleic acid. The muta form of the Cas9 protein that can comprise an amino acid tion can result in one or more of the plurality of nucleic change such as a deletion, insertion, Substitution, variant, acid-cleaving domains retaining the ability to cleave the mutation, fusion, chimera, or any combination thereof. non-complementary Strand of the target nucleic acid but 0189 In some embodiments, an actuator moiety com reducing its ability to cleave the complementary Strand of prises a “zinc finger nuclease” or “ZFN.” ZFNs refer to a the target nucleic acid. The mutation can result in one or fusion between a cleavage domain, such as a cleavage more of the plurality of nucleic acid-cleaving domains domain of FokI, and at least one Zinc finger motif (e.g., at lacking the ability to cleave the complementary strand and least 2, 3, 4, or 5 zinc finger motifs) which can bind the non-complementary strand of the target nucleic acid. The polynucleotides such as DNA and RNA. The heterodi residues to be mutated in a nuclease domain can correspond merization at certain positions in a polynucleotide of two to one or more catalytic residues of the nuclease. For individual ZFNs in certain orientation and spacing can lead example, residues in the wild type exemplary S. pyogenes to cleavage of the polynucleotide. For example, a ZFN Cas9 polypeptide such as Asp10. His840, Asn854 and binding to DNA can induce a double-strand break in the ASn856 can be mutated to inactivate one or more of the DNA. In order to allow two cleavage domains to dimerize plurality of nucleic acid-cleaving domains (e.g., nuclease and cleave DNA, two individual ZFNs can bind opposite domains). The residues to be mutated in a nuclease domain strands of DNA with their C-termini at a certain distance of a Cas protein can correspond to residues Asp10. His840, apart. In some cases, linker sequences between the Zinc Asn854 and Asn856 in the wild type S. pyogenes Cas9 finger domain and the cleavage domain can require the 5' polypeptide, for example, as determined by sequence and/or edge of each binding site to be separated by about 5-7 base structural alignment. pairs. In some cases, a cleavage domain is fused to the 0186. As non-limiting examples, residues D10, G12, C-terminus of each Zinc finger domain. Exemplary ZFNs G17, E762, H840, N854, N863, H982, H983, A984, D986, include, but are not limited to, those described in Urnov et and/or A987 (or the corresponding mutations of any of the al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Cas proteins) can be mutated. For example, e.g., D10A, Nat Methods, 2012, 9(8):805-7: U.S. Pat. Nos. 6,534,261; G12A, G17A, E762A, H840A, N854A, N863A, H982A, 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6.866,997: H983A, A984A, and/or D986A. Mutations other than ala 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; nine substitutions can be suitable. A D10A mutation can be 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; combined with one or more of H840A, N854A, or N856A 6,479,626; and U.S. Application Publication Nos. 2003/ mutations to produce a Cas9 protein Substantially lacking O232410 and 2009/0203140. DNA cleavage activity (e.g., a dead Cas9 protein). AH840A 0190. In some embodiments, an actuator moiety com mutation can be combined with one or more of D10A, prising a ZFN can generate a double-strand break in a target N854A, or N856A mutations to produce a site-directed polynucleotide, such as DNA. A double-strand break in polypeptide Substantially lacking DNA cleavage activity. A DNA can result in DNA break repair which allows for the N854A mutation can be combined with one or more of introduction of gene modification(s) (e.g., nucleic acid edit H840A, D10A, or N856A mutations to produce a site ing). DNA break repair can occur via non-homologous end directed polypeptide Substantially lacking DNA cleavage joining (NHEJ) or homology-directed repair (HDR). In activity. A N856A mutation can be combined with one or HDR, a donor DNA repair template that contains homology more of H840A, N854A, or D10A mutations to produce a arms flanking sites of the target DNA can be provided. In site-directed polypeptide substantially lacking DNA cleav Some embodiments, a ZFN is a Zinc finger nickase which age activity. induces site-specific single-strand DNA breaks or nicks, thus 0187. In some embodiments, a Cas protein is a Class 2 resulting in HDR. Descriptions of Zinc finger nickases are Cas protein. In some embodiments, a Cas protein is a type found, e.g., in Ramirez et al., Nucl Acids Res, 2012, 40(12): II Cas protein. In some embodiments, the Cas protein is a 5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33. In Cas9 protein, a modified version of a Cas9 protein, or Some embodiments, a ZFN binds a polynucleotide (e.g., derived from a Cas9 protein. For example, a Cas9 protein DNA and/or RNA) but is unable to cleave the polynucle lacking cleavage activity. In some embodiments, the Cas9 otide. protein is a Cas9 protein from S. pyogenes (e.g., SwissProt 0191 In some embodiments, the cleavage domain of an accession number Q99ZW2). In some embodiments, the actuator moiety comprising a ZFN comprises a modified Cas9 protein is a Cas9 from S. aureus (e.g., SwissProt form of a wild type cleavage domain. The modified form of accession number J7RUA5). In some embodiments, the the cleavage domain can comprise an amino acid change Cas9 protein is a modified version of a Cas9 protein from S. (e.g., deletion, insertion, or Substitution) that reduces the pyogenes or S. Aureus. In some embodiments, the Cas9 nucleic acid-cleaving activity of the cleavage domain. For protein is derived from a Cas9 protein from S. pyogenes or example, the modified form of the cleavage domain can have S. Aureus. For example, a S. pyogenes or S. Aureus Cas9 less than 90%, less than 80%, less than 70%, less than 60%, protein lacking cleavage activity. less than 50%, less than 40%, less than 30%, less than 20%, 0188 Cas9 can generally refer to a polypeptide with at less than 10%, less than 5%, or less than 1% of the nucleic least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, acid-cleaving activity of the wild-type cleavage domain. The 80%, 90%, 100% sequence identity and/or sequence simi modified form of the cleavage domain can have no substan larity to a wild type exemplary Cas9 polypeptide (e.g., Cas9 tial nucleic acid-cleaving activity. In some embodiments, the from S. pyogenes). Cas9 can refer to a polypeptide with at cleavage domain is enzymatically inactive. most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 0.192 In some embodiments, an actuator moiety com 80%, 90%, 100% sequence identity and/or sequence simi prises a “TALEN” or “TAL-effector nuclease.” TALENs US 2017/O 1983O8 A1 Jul. 13, 2017 26 refer to engineered transcription activator-like effector pairs in length. Meganucleases can be modular DNA-bind nucleases that generally contain a central domain of DNA ing nucleases such as any fusion protein comprising at least binding tandem repeats and a cleavage domain. TALENs one catalytic domain of an endonuclease and at least one can be produced by fusing a TAL effector DNA binding DNA binding domain or protein specifying a nucleic acid domain to a DNA cleavage domain. In some cases, a target sequence. The DNA-binding domain can contain at DNA-binding tandem repeat comprises 33-35 amino acids least one motif that recognizes single- or double-stranded in length and contains two hyperVariable amino acid resi DNA. The meganuclease can be monomeric or dimeric. In dues at positions 12 and 13 that can recognize at least one Some embodiments, the meganuclease is naturally-occurring specific DNA base pair. A transcription activator-like effec (found in nature) or wild-type, and in other instances, the tor (TALE) protein can be fused to a nuclease such as a meganuclease is non-natural, artificial, engineered, syn wild-type or mutated FokI endonuclease or the catalytic thetic, rationally designed, or man-made. In some embodi domain of FokI. Several mutations to FokI have been made ments, the meganuclease of the present disclosure includes for its use in TALENs, which, for example, improve cleav an I-CreI meganuclease, I-Ceul meganuclease, I-MSOI age specificity or activity. Such TALENs can be engineered meganuclease, I-Sce meganuclease, variants thereof, to bind any desired DNA sequence. TALENs can be used to derivatives thereof, and fragments thereof. Detailed descrip generate gene modifications (e.g., nucleic acid sequence tions of useful meganucleases and their application in gene editing) by creating a double-strand break in a target DNA editing are found, e.g., in Silva et al., Curr Gene Ther, 2011, sequence, which in turn, undergoes NHEJ or HDR. In some 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, cases, a single-stranded donor DNA repair template is pro 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, vided to promote HDR. Detailed descriptions of TALENs 111(11):4061-4066, and U.S. Pat. Nos. 7,842,489; 7,897, and their uses for gene editing are found, e.g., in U.S. Pat. 372: 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119, Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 361; 8,119,381; 8,124.36; and 8,129,134. 8,697.853; Scharenberg et al., Curr Gene Ther, 2013, 13(4): 0196. In some embodiments, the nuclease domain of a 29.1-303; Gaj et al., Nat Methods, 2012, 9(8):805-7: Beur meganuclease comprises a modified form of a wild type deley et al., Nat Commun, 2013, 4:1762; and Joung and nuclease domain. The modified form of the nuclease domain Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55. can comprise an amino acid change (e.g., deletion, insertion, 0193 In some embodiments, a TALEN is engineered for or Substitution) that reduces the nucleic acid-cleaving activ reduced nuclease activity. In some embodiments, the nucle ity of the nuclease domain. For example, the modified form ase domain of a TALEN comprises a modified form of a wild of the nuclease domain can have less than 90%, less than type nuclease domain. The modified form of the nuclease 80%, less than 70%, less than 60%, less than 50%, less than domain can comprise an amino acid change (e.g., deletion, 40%, less than 30%, less than 20%, less than 10%, less than insertion, or Substitution) that reduces the nucleic acid 5%, or less than 1% of the nucleic acid-cleaving activity of cleaving activity of the nuclease domain. For example, the the wild-type nuclease domain. The modified form of the modified form of the nuclease domain can have less than nuclease domain can have no substantial nucleic acid 90%, less than 80%, less than 70%, less than 60%, less than cleaving activity. In some embodiments, the nuclease 50%, less than 40%, less than 30%, less than 20%, less than domain is enzymatically inactive. In some embodiments, a 10%, less than 5%, or less than 1% of the nucleic acid meganuclease can bind DNA but cannot cleave the DNA. cleaving activity of the wild-type nuclease domain. The 0.197 In some embodiments, the actuator moiety is fused modified form of the nuclease domain can have no Substan to one or more transcription repressor domains, activator tial nucleic acid-cleaving activity. In some embodiments, the domains, epigenetic domains, recombinase domains, trans nuclease domain is enzymatically inactive. posase domains, flippase domains, nickase domains, or any 0194 In some embodiments, the transcription activator combination thereof. The activator domain can include one like effector (TALE) protein is fused to a domain that can or more tandem activation domains located at the carboxyl modulate transcription and does not comprise a nuclease. In terminus of the enzyme. In other cases, the actuator moiety Some embodiments, the transcription activator-like effector includes one or more tandem repressor domains located at (TALE) protein is designed to function as a transcriptional the carboxyl terminus of the protein. Non-limiting exem activator. In some embodiments, the transcription activator plary activation domains include GAL4, herpes simplex like effector (TALE) protein is designed to function as a activation domain VP16, VP64 (a tetramer of the herpes transcriptional repressor. For example, the DNA-binding simplex activation domain VP16), NF-KB p65 subunit, domain of the transcription activator-like effector (TALE) Epstein-Barr virus R transactivator (Rta) and are described protein can be fused (e.g., linked) to one or more transcrip in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. tional activation domains, or to one or more transcriptional Patent App. Publ. No. 2014.0068797. Non-limiting exem repression domains. Non-limiting examples of a transcrip plary repression domains include the KRAB (Krüppel tional activation domain include a herpes simplex VP16 associated box) domain of Kox 1, the Mad mSIN3 interac activation domain and a tetrameric repeat of the VP16 tion domain (SID), ERF repressor domain (ERD), and are activation domain, e.g., a VP64 activation domain. A non described in Chavez et al., Nat Methods, 2015, 12(4):326 limiting example of a transcriptional repression domain 328 and U.S. Patent App. Publ. No. 2014.0068797. An includes a Kirtippel-associated box domain. actuator moiety can also be fused to a heterologous poly 0.195. In some embodiments, an actuator moiety com peptide providing increased or decreased stability. The fused prises a meganuclease. Meganucleases generally refer to domain or heterologous polypeptide can be located at the rare-cutting endonucleases or homing endonucleases that N-terminus, the C-terminus, or internally within the actuator can be highly specific. Meganucleases can recognize DNA moiety. target sites ranging from at least 12 base pairs in length, e.g., 0198 An actuator moiety can comprise a heterologous from 12 to 40 base pairs, 12 to 50 base pairs, or 12 to 60 base polypeptide for ease of tracking or purification, Such as a US 2017/O 1983O8 A1 Jul. 13, 2017 27 fluorescent protein, a purification tag, or an epitope tag. the expression and/or activity of a target gene or edit a Examples of fluorescent proteins include green fluorescent nucleic acid sequence. FIGS. 4E-H show an analogous proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP. Emer system wherein receptor modification comprises a confor ald, Azami Green, Monomeric Azami Green, CopGFP. mational change. In some embodiments, the adaptor protein AceGFP, ZsCreen 1), yellow fluorescent proteins (e.g., YFP. is tethered to the membrane (e.g., as a membrane bound eYFP Citrine, Venus, YPet, PhiYFP, ZsYellow 1), blue fluo protein). rescent proteins (e.g. eBFP, eBFP2, AZurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins 0201 In some embodiments, the cleavage moiety only (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), cleaves the recognition site when in proximity to the cleav red fluorescent proteins (mKate, mKate2, mPlum, DsRed age recognition site. The cleavage recognition site can monomer, mCherry, mRFP1 DsRed-Express, DsRed2, comprise a polypeptide sequence that is a recognition DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, sequence of a protease. The cleavage moiety can comprise eqFP611, mRaspberry, mStrawberry, Jred), orange fluores protease activity which recognizes the polypeptide cent proteins (mCrange, mKO, Kusabira-Orange, Mono sequence. A cleavage moiety comprising protease activity meric Kusabira-Orange, mTangerine, tdTomato), and any can be a protease, or any derivative, variant or fragment other Suitable fluorescent protein. Examples of tags include thereof. A protease refers to any enzyme that performs glutathione-S-transferase (GST), chitin binding protein proteolysis, in which polypeptides are cleaved into Smaller polypeptides or amino acids. Various proteases are Suitable (CBP), maltose binding protein, thioredoxin (TRX), poly for use as a cleavage moiety. Some proteases can be highly (NANP), tandem affinity purification (TAP) tag, myc, AcV5, promiscuous such that a wide range of protein Substrates are AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, hydrolysed. Some proteases can be highly specific and only Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, cleave Substrates with a certain sequence, e.g., a cleavage T7, V5, VSV-G, histidine (His), biotin carboxyl carrier recognition sequence or peptide cleavage domain. In some protein (BCCP), and calmodulin. embodiments, the cleavage recognitions site comprises mul 0199 The cleavage recognition site of a GMP can be tiple cleavage recognition sequences, and each cleavage flanked by the antigen interacting domain and the actuator recognition sequence can be recognized by the same or moiety in some configurations of a chimeric receptor poly different cleavage moiety comprising protease activity (e.g., peptide. The actuator moiety can be released from the GMP protease). Sequence-specific proteases that can be used as by cleavage of the recognition site by a cleavage moiety. A cleavage moieties include, but are not limited to, Superfam cleavage moiety can recognize and/or cleave a cleavage ily CA proteases, e.g., families C1, C2, C6, C10, C12, C16, recognition site, for example, when in proximity to the C19, C28, C31, C32, C33, C39, C47, C51, C54, C58, C64, cleavage recognition site. A cleavage moiety can comprise a C65, C66, C67, C70, C71, C76, C78, C83, C85, C86, C87, polypeptide sequence. The cleavage moiety can form a C93, C96, C98, and C101, including papain (Carica portion of the chimeric adaptor polypeptide. The cleavage papaya), bromelain (Ananas comosus), cathepsin K (liver moiety can form the N-terminus, C-terminus, or an internal wort) and calpain (Homo sapiens); Superfamily CD pro portion of the chimeric adaptor polypeptide. In some teases, e.g., family C11, C13, C14, C25, C50, C80, and C84: embodiments, the cleavage moiety is complexed to the Such as caspase-1 (Rattus norvegicus) and separase (Sac chimeric adaptor polypeptide. The cleavage moiety can be charomyces cerevisiae); Superfamily CE protease, e.g., fam complexed to the N-terminus, C-terminus, or an internal ily C5, C48, C55, C57, C63, and C79 including adenain portion of the chimeric adaptor polypeptide. FIG. 3B shows (human adenovirus type 2); Superfamily CF proteases, e.g., an exemplary arrangement of the various components of a family C15 including pyroglutamyl-peptidase I (Bacillus subject system. The cleavage recognition site 302b of a amyloliquefaciens); Superfamily CL proteases, e.g., family GMP is flanked by the antigen interacting domain 301 and C60 and C82 including Sortase A (Staphylococcus aureus); the actuator moiety 302a, and the cleavage moiety 304 Superfamily CM proteases, e.g. family C18 including hepa forms a portion of a chimeric adaptor polypeptide 303. titis C virus peptidase 2 (hepatitis C virus); Superfamily CN (0200 FIGS. 4A-D illustrate schematically the release of proteases, e.g., family C9 including Sindbis virus-type nsP2 an actuator moiety from a GMP. FIG. 4A shows the binding peptidase (Sindbis virus); Superfamily CO proteases, e.g., of an antigen to a transmembrane chimeric receptor poly family C40 including dipeptidyl-peptidase VI (Lysinibacil peptide. The transmembrane chimeric receptor polypeptide lus sphaericus); Superfamily CP proteases, e.g., family C97 comprises an extracellular region having an antigen inter including DeSI-1 peptidase (Mus musculus); Superfamily PA acting domain 401 and an intracellular region comprising a proteases, e.g., family C3, C4, C24, C30, C37, C62, C74, GMP. The GMP includes an actuator moiety 402a linked to and C99 including TEV protease (Tobacco etch virus); a cleavage recognition site 402b. In response to antigen superfamily PB proteases, e.g., family C44, C45, C59, C69, binding, the receptor is modified by phosphorylation 403 in C89, and C95 including amidophosphoribosyltransferase the intracellular region of the receptor (FIG. 4B). Following precursor (homo sapiens); Superfamily PC proteases, fami receptor modification (e.g., phosphorylation), an adaptor lies C26, and C56 including Y-glutamyl hydrolase (Rattus protein comprising a receptor binding moiety is recruited to norvegicus); Superfamily PD proteases, e.g., family C46 the receptor as shown in FIG. 4C. The receptor comprises a including Hedgehog protein (Drosophila melanogaster); cleavage moiety 404; the cleavage moiety may be com Superfamily PE proteases, e.g., family P1 including Dmp A plexed with the adaptor or linked, for example by a peptide aminopeptidase (Ochrobactrum anthropi); others proteases, bond and/or peptide linker, to the receptor binding moiety. e.g., family C7, C8, C21, C23, C27, C36, C42, C53 and C75. When in proximity to the cleavage recognition site, the Additional proteases include serine proteases, e.g., those of cleavage moiety can cleave the recognition site to release the superfamily SB, e.g., families S8 and S53 including subtili actuator moiety from the GMP as shown in FIG. 4D. Upon sin (Bacillus licheniformis); those of Superfamily SC, e.g., release, the actuator moiety can enter the nucleus to regulate families S9, S10, S15, S28, S33, and S37 including prolyl US 2017/O 1983O8 A1 Jul. 13, 2017 28 oligopeptidase (Sus scrofa); those of Superfamily SE, e.g., 0202 In some embodiments, the cleavage recognition families S11, S12, and S13 including D-Ala-D-Ala pepti site comprises a cleavage recognition sequence (e.g., poly dase C (Escherichia coli); those of superfamily SF, e.g., peptide sequence or peptide cleavage domain) that is rec families S24 and S26 including signal peptidase I (Escheri ognized by a protease selected from the group consisting of chia coli); those of Superfamily SJ, e.g., families S16, S50, achromopeptidase, aminopeptidase, ancrod, angiotensin and S69 including Ion-A peptidase (Escherichia coli); those converting enzyme, bromelain, calpain, calpain I, calpain II, of Superfamily SK, e.g., families S14, S41, and S49 includ carboxypeptidase A, carboxypeptidase B, carboxypeptidase ing Clp protease (Escherichia coli); those of Superfamily G. carboxypeptidase P. carboxypeptidase W. carboxypepti SO, e.g., families S74 including Phage K1F endosialidase dase Y, caspase 1, caspase 2, caspase 3, caspase 4, caspase CIMCD self-cleaving protein (Enterobacteria phage K1F); 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, those of superfamily SP, e.g., family S59 including nucleo caspase 11, caspase 12, caspase 13, cathepsin B, cathepsin porin 145 (Homo sapiens); those of Superfamily SR, e.g., C, cathepsin D, cathepsin E, cathepsin G, cathepsin H, family S60 including Lactoferrin (Homo sapiens); those of cathepsin L, chymopapain, chymase, chymotrypsin, clostri superfamily SS, families S66 including murein tetrapepti pain, collagenase, complement C1r, complement C1s, dase LD-carboxypeptidase (Pseudomonas aeruginosa); complement Factor D, complement factor I, cucumisin, those of superfamily ST, e.g., families S54 including rhom dipeptidyl peptidase IV, elastase (leukocyte), elastase (pan boid-1 (Drosophila melanogaster); those of superfamily PA, creatic), endoproteinase Arg-C, endoproteinase Asp-N. e.g., families S1, S3, S6, S7, S29, S30, S31, S32, S39, S46, S55, S64, S65, and S75 including Chymotrypsin A (Bos endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, taurus); those of superfamily PB, e.g., families S45 and S63 factor Xa, ficin, furin, granzyme A, granzyme B, HIV including penicillin G acylase precursor (Escherichia coli); Protease, IGase, kallikrein tissue, leucine aminopeptidase those of superfamily PC, e.g., families S51 including dipep (general), leucine aminopeptidase (cytosol), leucine amino tidase E (Escherichia coli); those of superfamily PE, e.g., peptidase (microsomal), matrix metalloprotease, methionine families P1 including Dmp A aminopeptidase (Ochrobac aminopeptidase, neutrase, papain, pepsin, plasmin, proli trum anthropi); those unassigned, e.g., families S48, S62, dase, pronase E, prostate specific antigen, protease alkalo S68, S71, S72, S79, and S81 threonine proteases, e.g., those philic from Streptomyces griseus, protease from Aspergil of superfamily PB clan, e.g., families T1, T2, T3, and T6 lus, protease from Aspergillus Saitoi, protease from including archaean proteasome, B component (Thermo Aspergillus sojae, protease (B. licheniformis) (alkaline or plasma acidophilum); and those of Superfamily PE clan, alcalase), protease from Bacillus polymyxa, protease from e.g., family T5 including ornithine acetyltransferase (Sac Bacillus sp. protease from Rhizopus sp., protease S, protea charomyces cerevisiae); aspartic proteases, e.g., BACE1. Somes, proteinase from Aspergillus Oryzae, proteinase 3. BACE2; cathepsin D; cathepsin E., chymosin; napsin-A; proteinase A, proteinase K. protein C, pyroglutamate amino nepenthesin; pepsin; plasmepsin, presenilin; renin; and peptidase, rennin, rennin, Streptokinase, Subtilisin, ther HIV-1 protease, and metalloproteinases, e.g., exopeptidases, molysin, thrombin, tissue plasminogen activator, trypsin, metalloexopeptidases; endopeptidases, and metalloendo tryptase and urokinase. peptidases. A cleavage recognition sequence (e.g., polypep 0203 Table 1 lists exemplary proteases and associated tide sequence) can be recognized by any of the proteases recognition sequences that can be used in Systems of the disclosed herein. disclosure. TABLE 1. Exemplary proteases and associated recognition sequences Protease name Synonyms Recognition sequence Arg-C Arginyl peptidase, Endoproteinase Arg-C, Tissue R-X kallikrein Asp-N Endoproteinase Asp-N, Peptidyl-Asp x-D metalloendopeptidase Asp-N (N- Endoproteinase Asp-N, Peptidyl-Asp X-DE terminal Glu) metalloendopeptidase BNPS or 3-Bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole, W-x NCS. urea BNPS-skatol, N-chlorosuccinimidefurea Caspase-1 ICE, Interleukin-1B-Converting Enzyme FLWY-x-AHT-D- {DEKPQR} (SEQ ID NO: 61) Caspase-10 Flice2, Mch4 I-E-A-D-x (SEQ ID NO: 62) Caspase-2 Ich-1, Nedd2 D-V-A-D-DEKPQR} (SEQ ID NO: 63) or D-E-H- D-DEKPQR} (SEQ ID NO: 64) Caspase-3 Apopain, CPP32, Yama D-M-Q-D-DEKPQR}(SEQ ID NO: 65) or D-E-V-D- {DEKPQR} (SEQ ID NO: 66) Caspase-4 ICE(rel)II, Ich-2, TX L-E-V-D-DEKPQR} (SEQ US 2017/O 1983O8 A1 Jul. 13, 2017 29

TABLE 1-continued Exemplary proteases and associated recognition sequences

Protease name Synonyms Recognition sequence Caspase-5 ICE(rel)III, TY Caspase-6 Mch2 Caspase-7 CMH-1, ICE-LAP3, Mch-3 Caspase-8 FLICE, MASH, Mch5 Caspase-9 ICE-Lap6, Mchó Chymotrypsin Chymotrypsin (low specificity) Clostripain Clostridiopeptidase B CNBr. Cyanogen bromide CNBr (methyl Cyanogen bromide Cys) CNBr (with Cyanogen bromide acids) Enterokinase Enteropeptidase DE)(4)-K-x (SEQ ID NO: 74) Factor Xa. Coagulation factor Xa. AFGILTVM-DE-G-R-x (SEQ ID NO: 75) Formic acid Glu-C (AmAc Endoproteinase Glu-C, V8 protease, Glutamyl buffer) endopeptidase Glu-C (Phos Endoproteinase Glu-C, V8 protease, Glutamyl DE-x buffer) endopeptidase Granzyme B Cytotoxic T-lymphocyte proteinase 2, Granzyme-2, -E-P-D-x (SEQ ID NO: 76) GranzymeB, Lymphocyte protease, SECT, T-cell serine protease 1-3E HRV3C Human rhinovirus 3C protease, Picornain 3C, Protease brotease 3C Hydroxylamine Hydroxylammonium Odosobenzoic 2-Iodosobenzoic acid acid Lys-C Endoproteinase Lys-C, Lysyl endopeptidase Lys-N Endoproteinase Lys-N, Peptidyl-Lys metalloendopeptidase, Armiliaria mellea neutral proteinase Lys-N (Cys Endoproteinase Lys-N, Peptidyl-Lys modified) metalloendopeptidase, Armiliaria mellea neutral proteinase Mild acid hydrolysis NBS (long N-Bromosuccinimide exposure) NBS (short N-Bromosuccinimide exposure) NTCB 2-Nitro-5-thiocyanatobenzoic acid, 2-Nitro-5- hiocyanobenzoic acid Pancreatic Pancreatopeptidase E, Elastase-1 AGSV-x elastase Pepsin A Pepsin

Pepsin A (low Pepsin specificity)

Prolyl Prolyl oligopeptidase, Post-proline cleaving enzyme endopeptidase Proteinase K Endopeptidase K. Peptidase K TEV protease Tobacco etch virus protease, Nuclear-inclusion-a endopeptidase Thermolysin Thermophilic-bacterial protease Thrombin Factor IIa US 2017/O 1983O8 A1 Jul. 13, 2017 30

TABLE 1-continued Exemplary proteases and associated recognition sequences Protease name Synonyms Recognition sequence Trypsin Trypsin-1 x-KR-P} or W-K-P or M R-P But not: CD-K-D or C-K-HY) or C-R-K or R-R-HR) Trypsin (Arg K-P} blocked) Trypsin (Cys RKC-P} modified) Trypsin (Lys R-P} blocked)

0204 Proteases selected for use as cleavage moieties can be selected based on desired characteristics such as peptide bond selectivity, activity at certain pHs, molecular mass, etc. The properties of exemplary proteases are provided in Table 2. TABLE 2 Exemplary proteases and protease characteristics Peptide bond pH Molecular Accession Protease EC no. Class selectivity optimum mass (kDa) O. Endoproteinase

Trypsin (bovine) 3.4.21.4 serine P-P - (P1 = Lys, 8.0-9.0 23.5 POO76OS Arg) Chymotrypsin 3.4.21.1 serine P-P- (P = 7.5-8.5 25 POO766 (bovine) aromatic, P’ = nonspecific) Endoproteinase 3.424.33 metallo P1-Asp- (and -P1 6.0-8.0 27 op Asp-N cysteic acid) (Pseudomonas fragi) Endoproteinase serine -Arg-P1 8.0-8.5 30 Ila. Arg-C (mouse Submaxillary gland) Endoproteinase 3.4.21.19 serine -Glu-P'- (and 8.0 27 PO41888 Glu-C (V8 -Asp-P'-) (2) protease) (Staphylococci is attrelts) Endoproteinase 3.4.21.50 serine -Lys-P'- 8.0 30 NR 33 R S77957P Lys-C (Lysobacter enzymogenes) Pepsin (porcine) 3.4.23.1 aspartic P-P- (P = 2.0-4.0 34.5 POO791S hydrophobic preferred) Thermolysin 3.424.27 metallo P-P - (P = Leu, 7.0-9.0 37.5 POO800S (Bacilius Phe, Ile, Val, Met, thermoproteolyticus) Ala) Elastase (porcine) 3.4.21.36 serine P-P- (P = 7.8-8.5 25.9 POO772S uncharged, nonaromatic) Papain (Carica 3.4.22.2 cysteine P-P - (PI = Arg, 6.0-7.0 23 P00784. papaya) Lys preferred) Proteinase K 3.4.21.64 serine P-P- (P = 7.5-12.O 18.5 PO6873S (Tritirachium aromatic, album) hydrophobic preferred) Subtilisin 3.4.21.62 serine P-P- (P = 7...0-11.O 3OS 27.3. PO41898 (Bacilius subtilis) neutral acidic preferred) US 2017/O 1983O8 A1 Jul. 13, 2017 31

TABLE 2-continued Exemplary proteases and protease characteristics Peptide bond pH Molecular Accession Protease EC no. Class selectivity optimum mass (kDa) O. Clostripain 3.4.22.8 cysteine -Arg-P- (P = Pro 7.1-7.6 59 PO987OS (endoproteinase- preferred) Arg-C) (Clostridium histolyticum) Exopeptidase Carboxypeptidase 3.4.17.1 metallo P-P - (P cannot 7.0-8.0 34.5 POO73OS A (bovine) be Arg, Lys, Pro) Carboxypeptidase 3.4.17.2 metallo Pi-P'- (P = Lys, 7.0-9.0 34.6 POO732S B (porcine) Arg) Carboxypeptidase op serine P-P- 4.O-50 51 Ila. P (Penicillium (nonspecific) ianthinelium) Carboxypeptidase 3.4.16.5 serine P-P- S.S.-6.5 61 POO729S Y (yeast) (nonspecific) Cathepsin C 3.4.14.1 cysteine X-P-P- 5.5 210 Ila. (removes amino terminal dipeptide) Acylamino-acid- 3.4.19.1 serine Ac-P-P- (P = 7.5 8O-360 P19205 releasing enzyme Ser, Ala, Met (porcine) preferred) Pyroglutamate 3.4.19.3 cysteine P-P- (P = 7.0-9.0 70-80 Ila. aminopeptidase 5-oxoproline or (bovine) pyroglutamate)

0205. In some embodiments, the cleavage recognition example, an actuator moiety can be covalently linked (e.g., site comprises a first portion of an intein sequence that reacts at its N-terminus or C-terminus) via a peptide bond to a first with the second portion of the intein sequence to release the portion of the intein comprising a C-terminal intein. The actuator moiety. A heterologous split intein system can be actuator moiety-C-terminal intein fusion can be contacted used to facilitate release of the actuator moiety from the with a second portion of the intein sequence comprising an chimeric receptor polypeptide. The actuator moiety can be N-terminal intein. This contacting of the first and second covalently linked to the first portion of the intein sequence. portion of the inteins can result in a site-specific cleavage The actuator moiety can be linked via its N-terminus or (e.g., at a suitable site between the actuator moiety and the C-terminus to the first portion of the intein sequence. The C-terminal intein), thereby releasing the actuator moiety. second portion of the intein sequence can be a part of the chimeric adaptor polypeptide. The second portion of the 0207. In some embodiments, the cleavage recognition intein sequence can serve as a cleavage moiety. The first site comprises a disulfide bond. The disulfide bond can link portion or second portion of the intein sequence can be the the actuator moiety to the chimeric receptor polypeptide. N-terminal intein, the C-terminal intein, or any other suit The disulfide bond can be formed between one or more able portion of an intein that can facilitate release of the cysteines of the actuator moiety and the receptor. The actuator moiety. The intein sequences can be from any cysteines can be engineered into the actuator moiety or suitable source. The first and second portion can be from the receptor. The cysteines can be a part of the native or same or different sources (e.g., organism, protein). wild-type sequence. The cysteines can be present in a linker 0206. In an illustrative example shown in FIG. 13A, a peptide appended to the actuator moiety or the receptor. chimeric receptor polypeptide comprises an actuator moiety Cleavage of the disulfide bond can be facilitated by, for 1301 covalently linked (e.g., at its N-terminus or C-termi example, altering the redox conditions of the disulfide bond. nus) via a peptide bond to a first portion of the intein Alteration of the redox conditions can lead to reduction of sequence 1302, which comprises an N-terminal intein. The the disulfide bond to thiols and release of the actuator actuator moiety-N-terminal intein fusion can be contacted moiety. Cleavage of the disulfide bond can be facilitated by with a second portion of the intein sequence 1303 compris a cleavage moiety comprising a redox agent that can cata ing a C-terminal intein as shown in FIG. 13B, for example lyze reduction of the disulfide bond. The redox agent can be a second portion of the intein sequence linked to an adaptor an enzyme, or any derivative, variant or fragment thereof. polypeptide. This contacting of the first and second portion The enzyme can be an oxidoreductase. Examples of oxi of the intein sequences can result in a site specific cleavage doreductases include protein-disulfide reductase, thioredox (e.g., at a site between the actuator moiety and the N-ter ins, glutaredoxins, thiol disulfide oxidoreductases (e.g., minal intein) as shown in FIG. 13C, thereby releasing the DsbA, BdbA-D, MdbA, and SdbA), and glutathione disul actuator moiety as shown in FIG. 13D. In an alternative fide reductase. The redox agent can be from any Suitable configuration shown in FIGS. 13E-H, the actuator moiety is Source including prokaryotes and eukaryotes. Cofactors (e.g., linked and/or complexed to the adaptor polypeptide rather nicotinamide cofactors, flavins, and derivatives and analogs than the receptor polypeptide. In another illustrative thereof) can be supplied for optimal activity of the enzyme. US 2017/O 1983O8 A1 Jul. 13, 2017 32

0208. In an illustrative example shown in FIGS. 14A, a KFLKRR (SEQ ID NO. 53) of the mouse MX1 protein; the chimeric receptor polypeptide comprises an actuator moiety sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 1401 linked by disulfide bond. The disulfide bond can be 54) of the human poly(ADP-ribose) polymerase; and the cleaved by a cleavage moiety 1402 comprising an enzyme sequence RKCLQAGMNLEARKTKK (SEQID NO: 55) of Such as an oxidoreductase, for example an oxidoreductase the steroid hormone receptors (human) glucocorticoid. complexed and/or linked to an adaptor polypeptide as shown 0211. In some embodiments, a targeting sequence com in FIG. 14B. Cleaving of the disulfide bond can release the prises a membrane targeting peptide and directs a polypep actuator moiety as shown in FIG. 14C. The actuator moiety, tide to a plasma membrane or membrane of a cellular upon release, can translocate to a cell nucleus where it is organelle. A membrane-targeting sequence can provide for operable to regulate expression of a target polynucleotide transport of the chimeric transmembrane receptor polypep (e.g., gene expression) and/or activity or edit a nucleic acid tide to a cell surface membrane or other cellular membrane. sequence as shown in FIG. 14.D. FIGS. 14E-H illustrate an Molecules in association with cell membranes contain cer alternative configuration wherein the actuator moiety is tain regions that facilitate membrane association, and Such complexed and/or linked to the adaptor polypeptide and the regions can be incorporated into a membrane targeting cleavage moiety (e.g., oxidoreductase) is linked to the sequence. For example, Some proteins contain sequences at receptor. the N-terminus or C-terminus that are acylated, and these 0209. In some embodiments, the chimeric receptor poly acyl moieties facilitate membrane association. Such peptide comprises at least one targeting sequence which sequences can be recognized by acyltransferases and often directs transport of the receptor to a specific region of a cell. conform to a particular sequence motif. Certain acylation A targeting sequence can be used to direct transport of a motifs are capable of being modified with a single acyl polypeptide to which the targeting sequence is linked to a moiety (often followed by several positively charged resi specific region of a cell. For example, a targeting sequence dues (e.g. human c-Src) to improve association with anionic can direct the receptor to a cell nucleus utilizing a nuclear lipid head groups) and others are capable of being modified localization signal (NLS), outside of the nucleus (e.g., the with multiple acyl moieties. For example the N-terminal cytoplasm) utilizing a nuclear export signal (NES), the sequence of the protein tyrosine kinase Src can comprise a mitochondria, the endoplasmic reticulum (ER), the Golgi, single myristoyl moiety. Dual acylation regions are located chloroplasts, apoplasts, peroxisomes, plasma membrane, or within the N-terminal regions of certain protein kinases, membrane of various organelles of a cell. In some embodi Such as a Subset of Src family members (e.g., Yes, Fyn, Lck) ments, a targeting sequence comprises a nuclear export and G-protein alpha subunits. Such dual acylation regions signal (NES) and directs a polypeptide outside of a nucleus, often are located within the first eighteen amino acids of for example to the cytoplasm of a cell. A targeting sequence Such proteins, and conform to the sequence motif Met-Gly can direct a polypeptide to the cytoplasm utilizing various Cys-Xaa-Cys (SEQ ID NO: 56), where the Met is cleaved, nuclear export signals. Nuclear export signals are generally the Gly is N-acylated and one of the Cys residues is short amino acid sequences of hydrophobic residues (e.g., at S-acylated. The Gly often is myristoylated and a Cys can be least about 2, 3, 4, or 5 hydrophobic residues) that target a palmitoylated. Acylation regions conforming to the protein for export from the cell nucleus to the cytoplasm sequence motif Cys-Ala-Ala-Xaa (so called “CAAX through the nuclear pore complex using nuclear transport. boxes”), which can modified with C15 or C10 isoprenyl Not all NES substrates can be constitutively exported from moieties, from the C-terminus of G-protein gamma Subunits the nucleus. In some embodiments, a targeting sequence and other proteins also can be utilized. These and other comprises a nuclear localization signal (NLS, e.g., a SV40 acylation motifs include, for example, those discussed in NLS) and directs a polypeptide to a cell nucleus. A targeting Gauthier-Campbell et al., Molecular Biology of the Cell 15: sequence can direct a polypeptide to a cell nucleus utilizing 2205-2217 (2004); Glabati et al., Biochem. J.303:697-700 various nuclear localization signals (NLS). An NLS can be (1994) and Zlakine et al., J. Cell Science 110: 673-679 a monopartite sequence or a bipartite sequence. (1997), and can be incorporated in a targeting sequence to 0210. Non-limiting examples of NLSs include and NLS induce membrane localization. sequence derived from: the NLS of the SV40 virus large 0212. In certain embodiments, a native sequence from a T-antigen, having the amino acid sequence PKKKRKV protein containing an acylation motif is incorporated into a (SEQ ID NO: 40); the NLS from nucleoplasmin (e.g. the targeting sequence. For example, in Some embodiments, an nucleoplasmin bipartite NLS with the sequence KRPAAT N-terminal portion of Lck, Fyn or Yes or a G-protein alpha KKAGQAKKKK (SEQ ID NO: 41)); the c-myc NLS hav subunit, such as the first twenty-five N-terminal amino acids ing the amino acid sequence PAAKRVKLD (SEQ ID NO: or fewer from such proteins (e.g., about 5 to about 20 amino 42) or RQRRNELKRSP (SEQID NO:43); the hRNPA1 M9 acids, about 10 to about 19 amino acids, or about 15 to about NLS having the sequence NQSSNFGPMKGGNFGGRSS 19 amino acids of the native sequence with optional muta GPYGGGGQYFAKPRNQGGY (SEQ ID NO: 44); the tions), may be incorporated within the N-terminus of a Sequence RMRIZFKNKGKDTAELRRRRVEVSVEL chimeric polypeptide. In certain embodiments, a C-terminal RKAKKDEQILKRRNV (SEQ ID NO: 45) of the IBB sequence of about 25 amino acids or less from a G-protein domain from importin-alpha; the sequences VSRKRPRP gamma Subunit containing a CAAX box motif sequence (SEQID NO:46) and PPKKARED (SEQID NO: 47) of the (e.g., about 5 to about 20 amino acids, about 10 to about 18 myoma T protein; the sequence PQPKKKPL (SEQ ID NO: amino acids, or about 15 to about 18 amino acids of the 48) of human p53: the sequence SALKKKKKMAP(SEQID native sequence with optional mutations) can be linked to NO: 49) of mouse c-abl IV; the sequences DRLRR (SEQ ID the C-terminus of a chimeric polypeptide. NO: 50) and PKQKKRK (SEQID NO: 51) of the influenza 0213 Any membrane-targeting sequence can be virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 52) employed. In some embodiments, such sequences include, of the Hepatitis virus delta antigen; the sequence REKK but are not limited to myristoylation-targeting sequence, US 2017/O 1983O8 A1 Jul. 13, 2017 palmitoylation-targeting sequence, prenylation sequences 0218. In some cases, a targeting sequence is linked to the (i.e., farnesylation, geranyl-geranylation, CAAX Box), pro actuator moiety. Following release of the actuator moiety tein-protein interaction motifs or transmembrane sequences from the GMP (and receptor) by cleavage of the cleavage (utilizing signal peptides) from receptors. Examples include recognition site, the targeting sequence can direct the actua those discussed in, for example, ten Klooster, J. P. et al. tor moiety to a cellular location that is different from the Biology of the Cell (2007) 99, 1-12: Vincent, S., et al., receptor. For example, a chimeric transmembrane receptor Nature Biotechnology 21:936-40, 1098 (2003). can comprise a first targeting sequence directing the receptor 0214. Additional protein domains exist that can increase to a plasma membrane and the actuator moiety can sepa protein retention at various membranes. For example, an rately comprise a second targeting sequence directing local ~120 amino acid pleckstrin homology (PH) domain is found ization to a cell nucleus. Initially, the actuator moiety in over 200 human proteins that are typically involved in (forming a portion of the receptor) can be localized to a intracellular signaling. PH domains can bind various phos plasma membrane due to the first targeting sequence. Fol phatidylinositol (PI) lipids within membranes (e.g. PI(3,4, lowing release of the actuator moiety from the GMP by 5)-P3, PI(3,4)-P2, PI (4,5)-P2) and thus can play a key role cleavage of the cleavage recognition site, the actuator moi in recruiting proteins to different membrane or cellular ety can localize to a cell nucleus via targeting by the second compartments. Often the phosphorylation state of PI lipids targeting sequence. In some embodiments, the actuator is regulated, such as by PI-3 kinase or PTEN, and thus, moiety translocates to a cell nucleus after cleavage of the interaction of membranes with PH domains may not be as cleavage recognition sequence. stable as by acyl lipids. 0219 Binding of the chimeric adaptor polypeptide to a 0215. In some embodiments, a targeting sequence direct chimeric receptor polypeptide when the receptor has under ing a polypeptide to a cellular membrane can utilize a gone modification upon binding to an antigen can bring the membrane anchoring signal sequence. Various membrane cleavage moiety in proximity to the cleavage recognition anchoring sequences are available. For example, membrane site. Cleavage of the recognition site can release the actuator anchoring signal sequences of various membrane bound moiety from the GMP. Following release, the actuator proteins can be used. Sequences can include those from: 1) moiety is operable to complex with a target polynucleotide, class I integral membrane proteins such as IL-2 receptor for example in the cell cytoplasm or a cell nucleus. Com beta-chain and insulin receptor beta chain; 2) class II inte plexing of the actuator moiety with a target polynucleotide gral membrane proteins such as neutral endopeptidase; 3) can regulate the expression and/or activity of at least one type III proteins such as human cytochrome P450 NF25; and gene or edit a nucleic acid sequence. 4) type IV proteins such as human P-glycoprotein. 0220. In another exemplary configuration, the GMP 0216. In some embodiments, the chimeric receptor poly forms a portion of the chimeric adaptor polypeptide and the peptide is linked to a polypeptide folding domain which can cleavage moiety forms a portion of a chimeric receptor assist in protein folding. In some embodiments, an actuator polypeptide. A chimeric adaptor polypeptide of an exem moiety is linked to a cell-penetrating domain. For example, plary configuration can comprise (a) a receptor binding the cell-penetrating domain can be derived from the HIV-1 moiety that binds a receptor that has undergone modification TAT protein, the TLM cell-penetrating motif from human upon binding to an antigen; and (b) a gene modulating hepatitis B virus, MPG, Pep-1, VP22, a cell penetrating polypeptide (GMP) linked to the receptor binding moiety, peptide from Herpes simplex virus, or a polyarginine peptide wherein the GMP comprises an actuator moiety linked to a sequence. The cell-penetrating domain can be located at the cleavage recognition site; wherein (i) the cleavage recogni N-terminus, the C-terminus, or anywhere within the actuator tion site is cleavable by a cleavage moiety in response to moiety. receptor binding, and (ii) the actuator moiety is operable to 0217. The targeting sequence can be linked to any appro complex with a target polynucleotide in response to cleav priate region of the chimeric receptor polypeptide, for age of the cleavage recognition site. FIG. 6A shows an example at the N-terminus, the C-terminus, or in an internal exemplary chimeric adaptor polypeptide. A chimeric adaptor region of the receptor. In some embodiments, at least two polypeptide can comprise a receptor binding moiety 601 targeting sequences are linked to the receptor. In an exem linked to a GMP 602. A GMP can comprise an actuator plary chimeric receptor polypeptide shown in FIG. 5, a first moiety 602a linked to a cleavage recognition site 602b. targeting sequence 501 a can be linked to the extracellular 0221) A receptor binding moiety of a chimeric adaptor region of the receptor and a second targeting sequence 501b polypeptide can be any binding partner (e.g., protein) which can be linked to the intracellular region of the receptor, such can bind a receptor, or any derivative, variant or fragment as to the GMP. When a receptor is linked to multiple thereof. In some embodiments, an adaptor comprises a targeting sequences, for example targeting sequences binding partner of a receptor that is membrane-bound, or any directed to different locations of a cell, the final localization derivative, variant or fragment thereof. In some embodi of the receptor can be determined by the relative strengths of ments, an adaptor comprises a binding partner of a receptor, the targeting sequences. For example, a receptor having both or any derivative, variant or fragment thereof, that is not a targeting sequence comprising an NES and a targeting membrane-bound (e.g., intracellular or cytosolic). An adap sequence comprising an NLS can localize to the cytoplasm tor polynucleotide may comprise a receptor binding domain if the NES is stronger than NLS. Alternatively, if the NLS is of a signaling protein or other protein recruited to a receptor. stronger than the NES, the receptor can localize to the The chimeric adaptor polypeptide can be recruited to the nucleus even though both a nuclear localization signal and chimeric receptor polypeptide in response to receptor modi nuclear export signal are present on the receptor. A targeting fication, e.g., a conformational change, chemical modifica sequence can comprise multiple copies of for example, each tion, or combination thereof. A receptor may undergo recep a NLS and NES, to fine-tune the degree of the cellular tor modification in response to ligand binding. Receptors, or localization. any derivative, variant or fragment thereof, and binding US 2017/O 1983O8 A1 Jul. 13, 2017 34 partners (e.g., proteins), or any derivative, variant or frag complex, PIP5K1C, RAB8E, RAPGEF2, DDR1, PSEN1, ment thereof, can be selected so as to optimize the desired CDH1, CDC27, CTNNA1, and EGFR. level of recruitment of the adaptor polypeptide to the recep 0226. In some embodiments, a chimeric adaptor poly tOr. peptide comprises a molecule (e.g., protein) that is recruited 0222. In some embodiments, a chimeric adaptor poly to chimeric receptor polypeptide comprising a RTSK, or any peptide comprises a molecule (e.g., protein), or any deriva derivative, variant or fragment thereof. In some embodi tive, variant or fragment thereof, recruited to a Notch ments, a chimeric adaptor polypeptide comprises a protein, receptor when the Notch receptor is bound to a ligand. A or any derivative, variant or fragment thereof, selected from chimeric adaptor polypeptide can comprise a protein, any a SMAD family member including SMAD1, SMAD2, derivative, variant or fragment thereof, selected from the SMAD3, SMAD5, SMAD6, and SMAD7, and SMAD9 group consisting of presenilin-1 (PSEN1), nicastrin, anterior (sometimes referred to as SMAD8); the SMAD anchor for pharynx-defective 1 (APH-1), and presenilin enhancer 2 receptor activation (SARA); a SMURF protein (e.g., (PEN-2). SMURF1, SMURF2); and any derivative, variant or frag ment thereof. In some embodiments, a chimeric adaptor 0223) In some embodiments, a chimeric adaptor poly polypeptide comprises a molecule (e.g., protein) that is peptide comprises a molecule (e.g., protein), or any deriva recruited to chimeric receptor polypeptide comprising a tive, variant or fragment thereof, recruited to a GPCR when cytokine receptor, or any derivative, variant or fragment the GPCR is bound to a ligand (e.g., a ligand-bound GPCR thereof. In some embodiments, an adaptor polypeptide com that has undergone conformational and/or biochemical prises a gp130, CD131, CD132, or any derivative, variant or modification). A chimeric adaptor polypeptide can comprise fragment thereof. a protein, or any derivative, variant or fragment thereof, selected from the group consisting of: AKAP79 (AKAP5) 0227. In some embodiments, a chimeric adaptor poly and AKAP250 (AKAP12, gravin), arrestin (e.g., (3-arres peptide comprises a molecule recruited to a phosphorylated tin), ATBP50, calmodulin, DRIP78 (DNAJC14), Homer, RTK or RTSK, or a receptor phosphorylated by a non GASP1, GEC1 (GABARAPL1), INAD, JAK2, LARG (AR covalently associated intracellular kinase. The phosphory HGEF12), MAGI2, MAGI3, M10 MHC, MPP3, MRAP and lation of specific amino acid residues (e.g., tyrosine resi MRAP2, MUPP1 (MPDZ), neurochondrin, NHERF1 dues) within an activated receptor (e.g., a chimeric receptor (EBP50, SLC9A3R1), NHERF2 (SLC9A3R2), NINAA, polypeptide) can create binding sites for molecules Such as ODR4, p85, PDZ-RhoGEF (ARHGEF11), periplakin, PICK Src homology 2 (SH2) domain- and phosphotyrosine bind 1, PSD95, RACK1 (GNB2L1), RAMP1, RAMP2, RAMP3, ing (PTB) domain-containing proteins. In some embodi RanBP2, REEPs, RTPs, RTP4, Shank, SNX1, syntrophin, ments, an adaptor polypeptide comprises a protein contain spinophilin, TCTEXT1 (DYNLT1), and USP4. ing an SH2 domain, such as ABL1, ABL2, BCAR3, BLK, BLNK, BMX, BTK, CHN2, CISH, CRK, CRKL, CSK, 0224. In some embodiments, a chimeric adaptor poly DAPP1, EAT-2, FER FES, FGR, FRK, FYN, GADS, peptide comprises a molecule (e.g., protein), or any deriva GRAP, GRAP2, GRB10, GRB14, GRB2, GRB7, HCK, tive, variant or fragment thereof, that is recruited to an HSH2D, INPP5D, INPPL1, ITK, JAK2, LCK, LCP2, LYN, integrin receptor when the receptor is bound to a ligand. MATK, NCK1, NCK2, PIK3R1, PIK3R2, PIK3R3, PLCG1, Examples of adaptor proteins that are recruited to an integrin PLCG2, PTK6, PTPN11, PTPN6, RASA1, SAP, SH2B1, receptor include, but are not limited to, structural adaptor SH2B2, SH2B3, SH2D1A, SH2D1B, SH2D2A, SH2D3A, proteins, scaffolding adaptor proteins, and adaptor proteins SH2D3C, SH2D4A, SH2D4B, SH2D5, SH2D6, SH3BP2, having catalytic activity. In some embodiments, a chimeric SHB, SHC1, SHC2, SHC3, SHC4, SHD, SHE, SHP1, adaptor polypeptide comprises a protein, or any derivative, SHP2, SLA, SLA2, SOCS1, SOCS2, SOCS3, SOCS4, variant or fragment thereof, selected from talin, kindlin, SOCS5, SOCS6, SOCS7, SRC, SRMS, STAT1, STAT2, filamin and tensin. In some embodiments, a chimeric adap STAT3, STAT4, STAT5A, STAT5B, STAT6, SUPT6H, tor polypeptide comprises a protein, or any derivative, SYK, TEC, TENC1, TNS, TNS1, TNS3, TNS4, TXK, variant or fragment thereof, selected from paxillin and VAV1, VAV2, VAV3, YES1, ZAP70, or any derivative, kindlin. In some embodiments, a chimeric adaptor polypep variant or fragment thereof. In some embodiments, an tide comprises a protein, or any derivative, variant or adaptor polypeptide comprises a protein containing a PTB fragment thereof, selected from focal adhesion kinase domain, such as APBA1, APBA2, APBA3, EPS8, EPS8L1, (FAK), Src, and protein phosphatase 2A (PP2A). In some EPS8L2, EPS8L3, TENC1, TNS, TNS1, TNS3, TNS4, embodiments, a chimeric adaptor polypeptide comprises a DOK1, DOK2, DOK3, DOK4, DOK5, DOK6, DOK7, protein, or any derivative, variant or fragment thereof, FRS2, FRS3, IRS1, IRS2, IRS3, IRS4, SHC1, SHC2, SHC3, selected from RAB21, PTPN2, AUP1, BIN1, COL8A1, and SHC4, TLN1, TLN2, X11 a, or any derivative, variant or ITGB1. fragment thereof. 0225. In some embodiments, a chimeric adaptor poly 0228. In some embodiments, a chimeric adaptor poly peptide comprises a molecule (e.g., protein), or any deriva peptide comprises a protein that is recruited to a TNF tive, variant or fragment thereof, recruited to a cadherin receptor, or any derivative, variant or fragment thereof. Such receptor. The molecule may be recruited to the receptor as a proteins are sometimes referred to as TNR receptor associ result of a receptor modification (e.g., chemical modification ated factors or TRAFS and include TRAF1, TRAF2, e.g., phosphorylation, and/or conformational change). In TRAF3, TRAF4, TRAF5, TRAF6, and TRAF7. In some Some embodiments, a chimeric adaptor polypeptide com embodiments, a chimeric adaptor polypeptide comprises a prises a protein, or any derivative, variant or fragment receptor-interacting serine/threonine-protein kinase 1 (RIP1 thereof, selected from C-catenin, B-catenin, Y-catenin, or RIPK1) and receptor-interacting serine/threonine-protein catenin delta-1 (p120-catenin), AJAP1, CTNND1, kinase 3 (RIP3 or RIPK3), or any derivative, variant or DLGAP5, TBC1D2, LIMA1, CAV1, TRPV4, CTNNB1 fragment thereof. In some embodiments, a chimeric adaptor US 2017/O 1983O8 A1 Jul. 13, 2017

polypeptide comprises an adaptor protein that is recruited to from a DNA nuclease that can induce transcriptional acti a TNFR, such as Fas-associated protein with Dead Domain Vation or repression of a target DNA sequence. In some (FADD) and tumor necrosis factor receptor type-1 associ embodiments, the actuator moiety comprises a nuclease-null ated DEATH domain (TRADD) which binds TRAF2, or any RNA binding protein derived from a RNA nuclease that can derivative, variant or fragment thereof. induce transcriptional activation or repression of a target 0229. In some embodiments, a chimeric adaptor poly RNA sequence. In some embodiments, the actuator moiety peptide comprises a molecule (e.g., protein), or any deriva is a nucleic acid-guided actuator moiety. In some embodi tive, variant or fragment thereof, recruited to a phosphory ments, the actuator moiety is a DNA-guided actuator moiety. lated ITAM, for example an ITAM of a chimeric polypeptide In some embodiments, the actuator moiety is an RNA receptor comprising an immune receptor Such as a TCR. The guided actuator moiety. An actuator moiety can regulate phosphorylation of specific tyrosine residues within the expression or activity of a gene and/or edit a nucleic acid activated receptor can create binding sites for molecules sequence, whether exogenous or endogenous. For example, Such as Src homology 2 (SH2) domain- and phosphotyrosine an actuator moiety can comprise a Cas protein which lacks binding (PTB) domain-containing proteins. In some cleavage activity. embodiments, a chimeric adaptor polypeptide comprises 0231. Any suitable nuclease can be used in an actuator ABL1, ABL2, BCAR3, BLK, BLNK, BMX, BTK, CHN2, moiety. Suitable nucleases include, but are not limited to, CISH, CRK, CRKL, CSK, DAPP1, EAT-2, FER, FES, FGR, CRISPR-associated (Cas) proteins or Cas nucleases includ FRK, FYN, GADS, GRAP, GRAP2, GRB10, GRB14, ing type I CRISPR-associated (Cas) polypeptides, type II GRB2, GRB7, HCK, HSH2D, INPP5D, INPPL1, ITK, CRISPR-associated (Cas) polypeptides, type III CRISPR JAK2, LCK, LCP2, LYN, MATK, NCK1, NCK2, PIK3R1, associated (Cas) polypeptides, type IV CRISPR-associated PIK3R2, PIK3R3, PLCG1, PLCG2, PTK6, PTPN11, (Cas) polypeptides, type V CRISPR-associated (Cas) poly PTPN6, RASA1, SAP, SH2B1, SH2B2, SH2B3, SH2D1A, peptides, and type VI CRISPR-associated (Cas) polypep SH2D1B, SH2D2A, SH2D3A, SH2D3C, SH2D4A, tides; Zinc finger nucleases (ZFN); transcription activator SH2D4B, SH2D5, SH2D6, SH3BP2, SHB, SHC1, SHC2, like effector nucleases (TALEN); meganucleases; RNA SHC3, SHC4, SHD, SHE, SHP1, SHP2, SLA, SLA2, binding proteins (RBP); CRISPR-associated RNA binding SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, proteins; recombinases; flippases; transposases; Argonaute SRC, SRMS, STAT1, STAT2, STAT3, STAT4, STAT5A, (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), STATSB, STAT6, SUPT6H, SYK, TEC, TENC1, TNS, archaeal Argonaute (aAgo), and eukaryotic Argonaute TNS1, TNS3, TNS4, TXK, VAV1, VAV2, VAV3, YES1, (eAgo)); any derivative thereof, any variant thereof; and any ZAP70, or any derivative, variant or fragment thereof. In fragment thereof. In some embodiments, the actuator moiety Some embodiments, a chimeric adaptor polypeptide com comprises a Cas protein that forms a complex with a guide prises APBA1, APBA2, APBA3, EPS8, EPS8L1, EPS8L2, nucleic acid, such as a guide RNA. In some embodiments, EPS8L3, TENC1, TNS, TNS1, TNS3, TNS4, DOK1, the actuator moiety comprises a RNA-binding protein (RBP) DOK2, DOK3, DOK4, DOK5, DOK6, DOK7, FRS2, FRS3, optionally complexed with a guide nucleic acid, such as a IRS1, IRS2, IRS3, IRS4, SHC1, SHC2, SHC3, SHC4, guide RNA, which is able to form a complex with a Cas TLN1, TLN2, X11a, or any derivative, variant or fragment protein. thereof. 0232. In some embodiments, the actuator moiety com 0230. In some configurations, a chimeric adaptor poly prises a RNA-binding protein (RBP) optionally complexed peptide of a Subject system can comprise a gene modulating with a guide nucleic acid, such as a guide RNA, which is polypeptide (GMP). A GMP, as described elsewhere herein, able to form a complex with a Cas protein. FIG. 6B shows can comprise an actuator moiety linked to a cleavage an exemplary chimeric adaptor polypeptide in which the recognition site. The actuator moiety can comprise a nucle actuator moiety comprises an RNA-binding protein 600a ase (e.g., DNA nuclease and/or RNA nuclease), modified optionally complexed with a guide nucleic acid. Upon nuclease (e.g., DNA nuclease and/or RNA nuclease) that is release from the RNA-binding protein (RBP), for example nuclease-deficient or has reduced nuclease activity com by dissociation of the guide nucleic acid from the RBP or pared to a wild-type nuclease, a variant thereof, a derivative cleavage of the cleavage recognition site, the guide nucleic thereof, or a fragment thereofas described elsewhere herein. acid can form a complex with a Cas protein 600b which is The actuator moiety can regulate expression or activity of a operable to regulate expression of a target polynucleotide gene and/or edit the sequence of a nucleic acid (e.g., a gene (e.g., gene expression) and/or activity or edit a nucleic acid and/or gene product). In some embodiments, the actuator sequence. In some embodiments, the actuator moiety com moiety comprises a DNA nuclease Such as an engineered prises a nuclease-null DNA binding protein derived from a (e.g., programmable or targetable) DNA nuclease to induce DNA nuclease that can induce transcriptional activation or genome editing of a target DNA sequence. In some embodi repression of a target DNA sequence. In some embodiments, ments, the actuator moiety comprises a RNA nuclease Such the actuator moiety comprises a nuclease-null RNA binding as an engineered (e.g., programmable or targetable) RNA protein derived from a RNA nuclease that can induce nuclease to induce editing of a target RNA sequence. In transcriptional activation or repression of a target RNA Some embodiments, the actuator moiety has reduced or sequence. For example, an actuator moiety can comprise a minimal nuclease activity. An actuator moiety having Cas protein which lacks cleavage activity. reduced or minimal nuclease activity can regulate expres 0233. In some embodiments, the cleavage recognition sion and/or activity of a gene by physical obstruction of a site is flanked by the receptor binding moiety and the target polynucleotide or recruitment of additional factors actuator moiety. The actuator moiety can be released from effective to Suppress or enhance expression of the target the GMP and from the chimeric adaptor polypeptide by polynucleotide. In some embodiments, the actuator moiety cleavage of the recognition site by a cleavage moiety. The comprises a nuclease-null DNA binding protein derived cleavage moiety can recognize and/or cleave a cleavage US 2017/O 1983O8 A1 Jul. 13, 2017 36 recognition site, for example, when in proximity to the shown in FIG. 10B. A second adaptor polypeptide 1007 cleavage recognition site. A cleavage moiety can comprise a comprising a cleavage moiety 1006 is also recruited to the polypeptide sequence. The cleavage moiety, in some con modified receptor (FIG. 10C). The cleavage moiety may be figurations, forms a portion of the chimeric receptor poly complexed with the second adaptor polypeptide or linked, peptide. The cleavage moiety can form the N-terminus, for example by a peptide bond and/or peptide linker, to the C-terminus or an internal portion of the chimeric receptor adaptor. When in proximity to the cleavage recognition site, polypeptide. In some embodiments, the cleavage moiety is the cleavage moiety can cleave the recognition site to release complexed to the chimeric receptor polypeptide. The cleav the actuator moiety from the GMP as shown in FIG. 10D. age moiety can be complexed to the N-terminus, C-termi Upon release, the actuator moiety can enter the nucleus to nus, or an internal portion of the chimeric receptor poly regulate the expression and/or activity of a target gene or peptide. In an exemplary configuration shown in FIG. 7, the edit a nucleic acid sequence. FIGS. 10E-H show an analo cleavage recognition site 702b is flanked by the receptor gous system wherein receptor modification comprises a binding moiety 701 and the actuator moiety 702a, and the conformational change. In some embodiments, the chimeric cleavage moiety 706 forms a portion a chimeric receptor adaptor polypeptide is tethered to the membrane (e.g., as a polypeptide 705. membrane bound protein). In some embodiments, the sec 0234 FIGS. 8A-D illustrate schematically the release of ond adaptor polypeptide is tethered to the membrane (e.g., an actuator moiety from a GMP. FIG. 8A shows the binding as a membrane bound protein). of an antigen to a transmembrane chimeric receptor poly 0237 FIGS. 16A-D illustrate schematically the release of peptide. The transmembrane chimeric receptor polypeptide an actuator moiety in a system comprising a first membrane comprises an extracellular region having an antigen inter tethered adaptor and a second cytoplasmic adaptor. FIG. acting domain 805 and an intracellular region comprising a 16A shows the association of a first membrane-tethered cleavage moiety 806. The cleavage moiety can be com adaptor comprising a membrane tethering domain 1601a plexed with the receptor or linked, for example by a peptide (e.g., CAAX), a protease recognition site 1601b (e.g., TEV), bond and/or peptide linker, to the receptor. The GMP forms and an actuator moiety 1601c with a chimeric transmem a portion of the chimeric adaptor polypeptide. The GMP, brane receptor 1602. The chimeric transmembrane receptor linked to the receptor binding moiety 801, includes an can function as a scaffold and includes at least two adaptor actuator moiety 802a linked to a cleavage recognition site binding sites (e.g., EGFR or receptor tyrosine kinase 802b. In response to antigen binding, the receptor is modi (RTK)). One adaptor binding site can be associated with a fied by phosphorylation 803 in the intracellular region of the membrane-tethered adaptor as shown in FIG. 16B. The receptor (FIG. 8B). Following receptor modification (e.g., association of the membrane-tethered adaptor, in some phosphorylation), the chimeric adaptor polypeptide is cases, is dependent on antigen binding to the receptor. In recruited to the receptor as shown in FIG. 8C. The receptor Some systems, the membrane-tethered adaptor is located in comprises a cleavage moiety 806. When in proximity to the proximity to the receptor and association may not depend on cleavage recognition site, the cleavage moiety can cleave the antigen binding to the receptor. As shown in FIGS. 16B and recognition site to release the actuator moiety from the GMP 16C, antigen interaction with the receptor can conditionally as shown in FIG. 8D. Upon release, the actuator moiety can recruit a second adaptor protein comprising a cytoplasmic enter the nucleus to regulate the expression and/or activity of receptor binding moiety 1603a and protease 1603b, to the a target gene or edit a nucleic acid sequence. FIGS. 8E-H other adaptor binding site of the receptor. The second show an analogous system wherein receptor modification adaptor protein comprising the protease, when recruited to comprises a conformational change. In some embodiments, the transmembrane receptor, can cleave the protease recog the chimeric adaptor protein is tethered to the membrane nition site 1601b of the membrane-tethered molecule, (e.g., as a membrane bound protein). thereby releasing the actuator moiety 1601c as shown in 0235. In another configuration, the cleavage moiety is FIG. 16D. complexed to a second adaptor polypeptide which binds the 0238. In some embodiments, the cleavage moiety only chimeric receptor polypeptide when the receptor polypep cleaves at the recognition site when in proximity to the tide has undergone modification. An illustrative example is cleavage recognition site. In some embodiments, the cleav shown in FIG. 9. The cleavage recognition site 902b is age recognition site comprises a polypeptide sequence (e.g., flanked by the receptor binding moiety 901 and the actuator a peptide cleavage domain) that is a recognition sequence of moiety 902a, and the cleavage moiety 906 forms a portion a protease. The cleavage moiety can comprise protease a second adaptor polypeptide 907. activity which recognizes the polypeptide sequence. A cleav 0236 FIGS. 10A-D illustrate schematically the release of age moiety comprising protease activity can be a protease an actuator moiety from a GMP. FIG. 10A shows the binding including, but not limited to, any protease described else of an antigen to a transmembrane chimeric receptor poly where herein, or any derivative, variant or fragment thereof. peptide. The transmembrane chimeric receptor polypeptide In some embodiments, the cleavage recognition site com comprises an extracellular region having an antigen inter prises multiple cleavage recognition sequences, and each acting domain and an intracellular region. The GMP, com cleavage recognition sequence can be recognized by the prising an actuator moiety linked to a cleavage recognition same or different cleavage moiety comprising protease site, forms a portion of a chimeric adaptor polypeptide. The activity (e.g., protease). cleavage recognition site 1002b is flanked by the receptor 0239. In some embodiments, the cleavage recognition binding moiety 1001 and the actuator moiety 1002a. In site comprises a first portion of an intein sequence that reacts response to antigen binding, the receptor is modified by with the second portion of the intein sequence to release the phosphorylation 1003 in the intracellular region (FIG. 10B). actuator moiety. A heterologous split intein system can be Following receptor modification (e.g., phosphorylation), the used to facilitate release of the actuator moiety from the chimeric adaptor polypeptide is recruited to the receptor as chimeric adaptor polypeptide. The actuator moiety can be US 2017/O 1983O8 A1 Jul. 13, 2017 37 covalently linked to the first portion of the intein sequence. plasmic reticulum (ER), the Golgi, chloroplasts, apoplasts, The actuator moiety can be linked via its N-terminus or peroxisomes, plasma membrane, or membrane of various C-terminus to the first portion of the intein sequence. The organelles of a cell. In some embodiments, a targeting cleavage moiety can comprise the second portion of the sequence comprises a nuclear export signal (NES) and intein sequence. The first portion or second portion of the directs the chimeric adaptor polypeptide outside of a cell intein sequence can be the N-terminal intein, the C-terminal nucleus. In some embodiments, a targeting sequence com intein, or any other Suitable portion of an intein that can prises a nuclear localization signal (NLS) and directs the facilitate release of the actuator moiety. The intein sequences adaptor to a cell nucleus. A targeting sequence can direct the can be from any Suitable source. The first and second portion adaptor to a cell nucleus utilizing various nuclear localiza can be from the same or different sources (e.g., organism, tion signals (NLS). In some embodiments, a targeting protein). In an illustrative example, an actuator moiety can sequence comprises a membrane targeting sequence and be covalently linked (e.g., at its N-terminus or C-terminus) directs the adaptor to a plasma membrane or membrane of via a peptide bond to a first portion of the intein sequence, a cellular organelle. A targeting sequence can direct a which comprises an N-terminal intein. The actuator moiety polypeptide to a membrane utilizing a membrane anchoring N-terminal intein fusion can be contacted with a second signal sequence as previously described. Various membrane portion of the intein sequence comprising a C-terminal anchoring sequences are available. intein. This contacting of the first and second portion of the 0242. The targeting sequence can be linked to any appro intein sequences can result in a site specific cleavage (e.g., priate region of the chimeric adaptor polypeptide, for at a site between the actuator moiety and the N-terminal example at the N-terminus or the C-terminus of the poly intein), thereby releasing the actuator moiety. In another peptide or in an internal region of the adaptor. In some illustrative example, an actuator moiety can be covalently embodiments, at least two targeting sequences are linked to linked (e.g., at its N-terminus or C-terminus) via a peptide the adaptor. For example, as shown in FIG. 11, a first bond to a first portion of the intein comprising a C-terminal targeting sequence 1101 a can be linked to the receptor intein. The actuator moiety-C-terminal intein fusion can be binding moiety of the adaptor and a second targeting contacted with a second portion of the intein sequence sequence 1101b can be linked to the GMP of the adaptor, for comprising an N-terminal intein. This contacting of the first example to the actuator moiety. When an adaptor is linked and second portion of the inteins can result in a site-specific to multiple targeting sequences, for example targeting cleavage (e.g., at a suitable site between the actuator moiety sequences directed to different locations of a cell, the final and the C-terminal intein), thereby releasing the actuator localization of the adaptor can be determined by the relative moiety. strengths of the targeting sequences. For example, an adap 0240. In some embodiments, the cleavage recognition tor having both a targeting sequence comprising an NES and site comprises a disulfide bond. The disulfide bond can link a targeting sequence comprising an NLS can localize to the the actuator moiety to the receptor binding moiety in a cytosol if the NES is stronger than the NLS. Alternatively, chimeric adaptor polypeptide. The disulfide bond can be if the NLS is stronger than the NES, the adaptor can localize formed between one or more cysteines of the actuator to the nucleus even though both a nuclear localization signal moiety and the receptor binding moiety. The cysteines can and nuclear export signal are present on the adaptor. A be engineered into the actuator moiety or receptor binding targeting sequence can comprise multiple copies of for moiety. The cysteines can be a part of the native or wild-type example, each a NLS and NES, to fine-tune the degree of the sequence of the actuator moiety or receptor binding moiety. cellular localization. The cysteines can be present in a linker peptide appended to 0243 In some cases, a targeting sequence is linked to the the actuator moiety or the receptor binding moiety. Cleavage actuator moiety. Following release of the actuator moiety of the disulfide bond can be facilitated by, for example, from the GMP (and adaptor) by cleavage of the cleavage altering the redox conditions of the disulfide bond. Altera recognition site, the targeting sequence can direct the actua tion of the redox conditions can lead to reduction of the tor moiety to a cellular location that is different from the disulfide bond to thiols and release of the actuator moiety. adaptor. For example, a chimeric adaptor polypeptide can Cleavage of the disulfide bond can be facilitated by a comprise a first targeting sequence directing the adaptor to cleavage moiety comprising a redox agent that can lead to the cell cytoplasm and the actuator moiety can separately reduction of the disulfide bond. The redox agent can be an comprise a second targeting sequence directing localization enzyme, or any derivative, variant or fragment thereof. The to a cell nucleus. Initially, the actuator moiety (forming a enzyme can be an oxidoreductase. Examples of oxidoreduc portion of the adaptor) can be localized to the cell cytoplasm tases include protein-disulfide reductase, thioredoxins, glu due to the first targeting sequence. Following release of the taredoxins, thiol disulfide oxidoreductases (e.g., DsbA, actuator moiety from the GMP by cleavage of the cleavage BdbA-D, MdbA, SdbA), and glutathione disulfide reduc recognition site, the actuator moiety can localize to a cell tase. The redox agent can be from any suitable source nucleus via targeting by the second targeting sequence. In including prokaryotes and eukaryotes. Cofactors (e.g., nico Some embodiments, the actuator moiety translocates to a cell tinamide cofactors, flavins, and derivatives and analogs nucleus after cleavage of the cleavage recognition sequence. thereof) can be supplied for optimal activity of the enzyme. 0244. In some embodiments, a targeting sequence com 0241. In some embodiments, the chimeric adaptor poly prises a membrane targeting peptide and directs a polypep peptide comprises at least one targeting sequence which tide to a plasma membrane or membrane of a cellular directs transport of the adaptor to a specific region of a cell. organelle. A membrane-targeting sequence can provide for For example, a targeting sequence can direct the adaptor to transport of the chimeric transmembrane receptor polypep a cell nucleus utilizing a nuclear localization signal (NLS), tide to a cell surface membrane or other cellular membrane. outside of a cell nucleus (e.g., to the cytoplasm) utilizing a Any suitable membrane target sequence previously nuclear export signal (NES), the mitochondria, the endo described herein may be used. US 2017/O 1983O8 A1 Jul. 13, 2017

0245. In some embodiments, the chimeric adaptor poly transcription factor I (COUP-TFI), chicken ovalbumin peptide is linked to a polypeptide folding domain which can upstream promoter-transcription factor II (COUP-TFII), assist in protein folding. In some embodiments, an actuator V-erbA-related (EAR-2), estrogen receptor-O. (ERC.), estro moiety can be linked to a cell-penetrating domain. For gen receptor-fi (ERB), estrogen-related receptor-O. (ERRC), example, the cell-penetrating domain can be derived from estrogen-related receptor-B (ERRB), estrogen-related recep the HIV-1 TAT protein, the TLM cell-penetrating motif from tor-Y (ERRY), glucocorticoid receptor (GR), mineralocorti human hepatitis B virus, MPG, Pep-1, VP22, a cell pen coid receptor (MR), progesterone receptor (PR), androgen etrating peptide from Herpes simplex virus, or a polyargi receptor (AR), nerve growth factor IB (NGFIB), nuclear nine peptide sequence. The cell-penetrating domain can be receptor related 1 (NURR1), neuron-derived orphan recep located at the N-terminus, the C-terminus, or anywhere tor 1 (NOR1), steroidogenic factor 1 (SF1), liver receptor within the actuator moiety. homolog-1 (LRH-1), and germ cell nuclear factor (GCNF). 0246 The actuator moiety of a subject system, upon 0249. A chimeric intracellular receptor comprising a release from a chimeric adaptor polypeptide or chimeric nuclear receptor, or any derivative, variant or fragment receptor polypeptide, can bind to a target polynucleotide to thereof, can bind an antigen comprising any suitable ligand regulate expression and/or activity of the target polynucle of a nuclear receptor, or any derivative, variant or fragment otide by physical obstruction of the target polynucleotide or thereof. Non-limiting examples of ligands of nuclear recep recruitment of additional factors effective to suppress or tors include thyroid hormone, Vitamin A and related com enhance expression of the target polynucleotide. In some pounds, fatty acids, prostaglandins, heme, cholesterol, embodiments, the actuator moiety comprises a transcrip ATRA, oxysterols, vitamin D, Xenobiotics, androstane, ret tional activator effective to increase expression of the target inoids, estrogens, cortisol, aldosterone, progesterone, testos polynucleotide. The actuator moiety can comprise a tran terone, and phosphatidylinositols. In some embodiments, the scriptional repressor effective to decrease expression of the antigen is a hormone. target polynucleotide. In some embodiments, the target 0250 FIGS. 12A-C illustrates schematically a system polynucleotide comprises genomic DNA. In some embodi comprising an exemplary intracellular receptor comprising a ments, the target polynucleotide comprises a region of a nuclear receptor. The system includes a receptor 1200 com plasmid, for example a plasmid carrying an exogenous gene. prising an actuator moiety 1201. In the absence of a ligand In some embodiments, the target polynucleotide comprises binding to the nuclear receptor, the receptor can be seques RNA, for example mRNA. In some embodiments, the target tered in a certain compartment of a cell, for example the polynucleotide comprises an endogenous gene or gene prod cytoplasm, by interaction with a binding protein 1202 as uct. The actuator moiety can include one or more copies of shown in FIG. 12A. Upon binding of a ligand 1203 to the a nuclear localization signal that allows the actuator to intracellular receptor as shown in FIG. 12B, the receptor can translocate into the nucleus upon cleavage from the GMP. dissociate from the binding protein 1202 and translocate to 0247. In some aspects, the chimeric receptor polypeptide the nucleus. The actuator moiety 1201 which is complexed is a chimeric intracellular receptor. An exemplary chimeric and/or linked to the receptor enters the nucleus with the intracellular receptor comprises (a) an antigen interacting receptor where it is operable to regulate expression of a domain that specifically binds an antigen, and (b) an actuator target polynucleotide (e.g., gene expression) and/or activity moiety linked to the antigen interacting domain. In some or edit a nucleic acid sequence. embodiments, (i) the chimeric intracellular receptor is modi 0251 The actuator moiety can comprise a nuclease (e.g., fied in response to antigen binding, (ii) the chimeric intra DNA nuclease and/or RNA nuclease), modified nuclease cellular receptor polypeptide translocates to a nucleus of a (e.g., DNA nuclease and/or RNA nuclease) that is nuclease cell in response to modification, and (iii) the actuator moiety deficient or has reduced nuclease activity compared to a complexes with a target polynucleotide in the nucleus. wild-type nuclease, a variant thereof, a derivative thereof, or 0248. In some embodiments, a chimeric intracellular a fragment thereof as described elsewhere herein. The receptor is a nuclear receptor. For example, a chimeric actuator moiety can regulate expression or activity of a gene intracellular receptor polypeptide can comprise a nuclear and/or edit the sequence of a nucleic acid (e.g., a gene and/or receptor, or any derivative, variant or fragment thereof, gene product). In some embodiments, the actuator moiety selected from a thyroid hormone receptor C. (TRC), thyroid comprises a DNA nuclease Such as an engineered (e.g., hormone receptor B (TRB), retinoic acid receptor-O. (RAR programmable or targetable) DNA nuclease to induce C.), retinoic acid receptor-B (RAR-3), retinoic acid recep genome editing of a target DNA sequence. In some embodi tor-Y (RAR-Y), peroxisome proliferator-activated receptor-C. ments, the actuator moiety comprises a RNA nuclease Such (PPARC), peroxisome proliferator-activated receptor-B/8 as an engineered (e.g., programmable or targetable) RNA (PPAR-B/ö), peroxisome proliferator-activated receptor-y nuclease to induce editing of a target RNA sequence. In (PPARY), Rev-ErbAC., Rev-Erb AB, RAR-related orphan Some embodiments, the actuator moiety has reduced or receptor-O. (RORC.), RAR-related orphan receptor-f minimal nuclease activity. An actuator moiety having (RORB), RAR-related orphan receptor-Y (RORY), Liver X reduced or minimal nuclease activity can regulate expres receptor-C., Liver X receptor-fi, Farnesoid X receptor, Farne sion and/or activity of a gene by physical obstruction of a soid X receptor-B, Vitamin D receptor, Pregnane X receptor, target polynucleotide or recruitment of additional factors constitutive adrostane receptor, hepatocyte nuclear factor effective to Suppress or enhance expression of the target 4-C. (HNF4C), hepatocyte nuclear factor-4-O. (HNF4y), ret polynucleotide. In some embodiments, the actuator moiety inoid X receptor-O. (RXRC.), retinoid X receptor-fi (RXRB), comprises a nuclease-null DNA binding protein derived retinoid X receptor-Y (RXRY), testicular receptor 2 (TR2), from a DNA nuclease that can induce transcriptional acti testicular receptor 4 (TR4), homologue of the Drosophila Vation or repression of a target DNA sequence. In some tailless gene (TLX), photoreceptor cell-specific nuclear embodiments, the actuator moiety comprises a nuclease-null receptor (PNR), chicken ovalbumin upstream promoter RNA binding protein derived from a RNA nuclease that can US 2017/O 1983O8 A1 Jul. 13, 2017 39 induce transcriptional activation or repression of a target inside of an organism. A cell can be an organism. A cell can RNA sequence. In some embodiments, the actuator moiety be a cell in a cell culture. A cell can be one of a collection is a nucleic acid-guided actuator moiety. In some embodi of cells. A cell can be a mammalian cell or derived from a ments, the actuator moiety is a DNA-guided actuator moiety. mammalian cell. A cell can be a rodent cell or derived from In some embodiments, the actuator moiety is an RNA a rodent cell. A cell can be a human cell or derived from a guided actuator moiety. An actuator moiety can regulate human cell. A cell can be a prokaryotic cell or derived from expression or activity of a gene and/or edit a nucleic acid a prokaryotic cell. A cell can be a bacterial cell or can be sequence, whether exogenous or endogenous. derived from a bacterial cell. A cell can be an archaeal cell 0252) Any suitable nuclease can be used in an actuator or derived from an archaeal cell. A cell can be a eukaryotic moiety. Suitable nucleases include, but are not limited to, cell or derived from a eukaryotic cell. A cell can be a CRISPR-associated (Cas) proteins or Cas nucleases includ pluripotent stem cell. A cell can be a plant cell or derived ing type I CRISPR-associated (Cas) polypeptides, type II from a plant cell. A cell can be an animal cell or derived from CRISPR-associated (Cas) polypeptides, type III CRISPR an animal cell. A cell can be an invertebrate cell or derived associated (Cas) polypeptides, type IV CRISPR-associated from an invertebrate cell. A cell can be a vertebrate cell or (Cas) polypeptides, type V CRISPR-associated (Cas) poly derived from a vertebrate cell. A cell can be a microbe cell peptides, and type VI CRISPR-associated (Cas) polypep or derived from a microbe cell. A cell can be a fungi cell or tides; Zinc finger nucleases (ZFN); transcription activator derived from a fungicell. A cell can be from a specific organ like effector nucleases (TALEN); meganucleases; RNA or tissue. binding proteins (RBP); CRISPR-associated RNA binding 0257. A cell can be a stem cell or progenitor cell. Cells proteins; recombinases; flippases; transposases; Argonaute can include stem cells (e.g., adult stem cells, embryonic proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argo stem cells, iPS cells) and progenitor cells (e.g., cardiac naute (aAgo), and eukaryotic Argonaute (eAgo)); any progenitor cells, neural progenitor cells, etc.). Cells can derivative thereof any variant thereof and any fragment include mammalian stem cells and progenitor cells, includ thereof. In some embodiments, the actuator moiety com ing rodent stem cells, rodent progenitor cells, human stem prises a Cas protein that forms a complex with a guide cells, human progenitor cells, etc. Clonal cells can comprise nucleic acid, Such as a guide RNA. the progeny of a cell. A cell can comprise a target nucleic 0253) The actuator moiety of an intracellular receptor, acid. A cell can be in a living organism. A cell can be a upon translocation to a cell nucleus (e.g., with the intracel genetically modified cell. A cell can be a host cell. lular receptor), can bind to a target polynucleotide to regu (0258. A cell can be a totipotent stem cell, however, in late expression and/or activity of the target polynucleotide some embodiments of this disclosure, the term "cell' may be by physical obstruction of the target polynucleotide or used but may not refer to a totipotent stem cell. A cell can recruitment of additional factors effective to suppress or be a plant cell, but in some embodiments of this disclosure, enhance expression of the target polynucleotide. In some the term "cell' may be used but may not refer to a plant cell. embodiments, the actuator moiety comprises a transcrip A cell can be a pluripotent cell. For example, a cell can be tional activator effective to increase expression of the target a pluripotent hematopoietic cell that can differentiate into polynucleotide. The actuator moiety can comprise a tran other cells in the hematopoietic cell lineage but may not be scriptional repressor effective to decrease expression of the able to differentiate into any other non-hematopoetic cell. A target polynucleotide. cell may be able to develop into a whole organism. A cell 0254. In some embodiments, the target polynucleotide may or may not be able to develop into a whole organism. comprises genomic DNA. In some embodiments, the target A cell may be a whole organism. polynucleotide comprises a region of a plasmid, for example 0259. A cell can be a primary cell. For example, cultures a plasmid carrying an exogenous gene. In some embodi of primary cells can be passaged 0 times, 1 time, 2 times, 4 ments, the target polynucleotide comprises RNA, for times, 5 times, 10 times, 15 times or more. Cells can be example mRNA. In some embodiments, the target poly unicellular organisms. Cells can be grown in culture. nucleotide comprises an endogenous gene or gene product. 0260 A cell can be a diseased cell. A diseased cell can 0255 In some cases, a targeting sequence is linked to the have altered metabolic, gene expression, and/or morpho intracellular receptor. For example, a targeting sequence can logic features. A diseased cell can be a cancer cell, a diabetic direct the receptor to a cell nucleus utilizing a nuclear cell, and a apoptotic cell. A diseased cell can be a cell from localization signal (NLS), outside of a cell nucleus (e.g., to a diseased subject. Exemplary diseases can include blood the cytoplasm) utilizing a nuclear export signal (NES), the disorders, cancers, metabolic disorders, eye disorders, organ mitochondria, the endoplasmic reticulum (ER), the Golgi, disorders, musculoskeletal disorders, cardiac disease, and chloroplasts, apoplasts, or peroxisomes. In some embodi the like. ments, a targeting sequence comprises a nuclear export 0261) If the cells are primary cells, they may be harvested signal (NES) and directs the receptor outside of a cell from an individual by any method. For example, leukocytes nucleus. In some embodiments, a targeting sequence com may be harvested by apheresis, leukocytapheresis, density prises a nuclear localization signal (NLS) and directs the gradient separation, etc. Cells from tissues such as skin, receptor to a cell nucleus. A targeting sequence can direct the muscle, bone marrow, spleen, liver, pancreas, lung, intestine, receptor to a cell nucleus utilizing various nuclear localiza stomach, etc. can be harvested by biopsy. An appropriate tion signals (NLS). In some embodiments, the chimeric Solution may be used for dispersion or Suspension of the intracellular receptor is linked to a polypeptide folding harvested cells. Such solution can generally be a balanced domain which can assist in protein folding. salt Solution, (e.g. normal saline, phosphate-buffered saline 0256 A subject system can be introduced into a variety of (PBS), Hank's balanced salt solution, etc.), conveniently cells. A cell can be in vitro. A cell can be in vivo. A cell can supplemented with fetal calf serum or other naturally occur be ex vivo. A cell can be an isolated cell. A cell can be a cell ring factors, in conjunction with an acceptable buffer at low US 2017/O 1983O8 A1 Jul. 13, 2017 40 concentration. Buffers can include HEPES, phosphate buf Sweat gland cell (odoriferous secretion, sex -hormone sen fers, lactate buffers, etc. Cells may be used immediately, or sitive), Gland of Moll cell in eyelid (specialized sweat they may be stored (e.g., by freezing). Frozen cells can be gland), Sebaceous gland cell (lipid-rich sebum secretion), thawed and can be capable of being reused. Cells can be Bowman's gland cell in nose (washes olfactory epithelium), frozen in a DMSO, serum, medium buffer (e.g., 10% Brunner's gland cell in duodenum (enzymes and alkaline DMSO, 50% serum, 40% buffered medium), and/or some mucus), Seminal vesicle cell (secretes seminal fluid com other such common Solution used to preserve cells at freez ponents, including fructose for Swimming sperm), Prostate ing temperatures. gland cell (secretes seminal fluid components), Bulboure 0262. Non-limiting examples of cells in which a subject thral gland cell (mucus secretion), Bartholin's gland cell system can be utilized include, but are not limited to, (vaginal lubricant secretion), Gland of Littre cell (mucus lymphoid cells, such as B cell, T cell (Cytotoxic T cell, secretion), Uterus endometrium cell (carbohydrate secre Natural Killer T cell, Regulatory T cell, T helper cell), tion), Isolated goblet cell of respiratory and digestive tracts Natural killer cell, cytokine induced killer (CIK) cells (see (mucus secretion), Stomach lining mucous cell (mucus e.g. US20080241194); myeloid cells, such as granulocytes secretion), Gastric gland Zymogenic cell (pepsinogen secre (Basophil granulocyte, Eosinophil granulocyte, Neutrophil tion), Gastric gland oxyntic cell (hydrochloric acid secre granulocyte/Hypersegmented neutrophil), Monocyte/Mac tion), Pancreatic acinar cell (bicarbonate and digestive rophage, Red blood cell (Reticulocyte), Mast cell. Throm enzyme secretion), Paneth cell of Small intestine (lysozyme bocyte/Megakaryocyte, Dendritic cell; cells from the endo secretion). Type II pneumocyte of lung (Surfactant Secre crine system, including thyroid (Thyroid epithelial cell, tion), Clara cell of lung, Hormone secreting cells, Anterior Parafollicular cell), parathyroid (Parathyroid chief cell, pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealo Gonadotropes, Corticotropes, Intermediate pituitary cell, cyte) cells; cells of the nervous system, including glial cells Magnocellular neurosecretory cells, Gut and respiratory (Astrocyte, Microglia), Magnocellular neurosecretory cell, tract cells, Thyroid gland cells, thyroid epithelial cell, para Stellate cell, Boettcher cell, and pituitary (Gonadotrope, follicular cell, Parathyroid gland cells, Parathyroid chief Corticotrope, Thyrotrope, Somatotrope, Lactotroph); cells cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley of the Respiratory system, including Pneumocyte (Type I dig cell of testes, Theca interna cell of ovarian follicle, pneumocyte, Type II pneumocyte), Clara cell, Goblet cell, Corpus luteum cell of ruptured ovarian follicle, Granulosa Dust cell; cells of the circulatory system, including Myo lutein cells, Theca lutein cells, Juxtaglomerular cell (renin cardiocyte, Pericyte; cells of the digestive system, including secretion), Macula densa cell of kidney, Metabolism and stomach (Gastric chief cell, Parietal cell), Goblet cell, storage cells, Barrier function cells (Lung, Gut, Exocrine Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S Glands and Urogenital Tract), Kidney, Type I pneumocyte cells; enteroendocrine cells, including enterochromaffm cell, (lining air space of lung), Pancreatic duct cell (centroacinar APUD cell, liver (Hepatocyte, Kupffer cell), Cartilage/bone/ cell), Nonstriated duct cell (of Sweat gland, salivary gland, muscle; bone cells, including Osteoblast, Osteocyte, Osteo mammary gland, etc.), Duct cell (of seminal vesicle, prostate clast, teeth (Cementoblast, Ameloblast); cartilage cells, gland, etc.), Epithelial cells lining closed internal body including Chondroblast, Chondrocyte; skin cells, including cavities, Ciliated cells with propulsive function, Extracellu Trichocyte, Keratinocyte, Melanocyte (Nevus cell); muscle lar matrix secretion cells, Contractile cells; Skeletal muscle cells, including Myocyte, urinary system cells, including cells, stem cell, Heart muscle cells, Blood and immune Podocyte, Juxtaglomerular cell, Intraglomerular mesangial system cells, Erythrocyte (red blood cell), Megakaryocyte cell/Extraglomerular mesangial cell, Kidney proximal (platelet precursor), Monocyte, Connective tissue macro tubule brush border cell, Macula densa cell; reproductive phage (various types), Epidermal Langerhans cell, Osteo system cells, including Spermatozoon, Sertoli cell, Leydig clast (in bone), Dendritic cell (in lymphoid tissues), Micro cell, Ovum; and other cells, including Adipocyte, Fibroblast, glial cell (in central nervous system), Neutrophil Tendon cell, Epidermal keratinocyte (differentiating epider granulocyte, Eosinophil granulocyte, Basophil granulocyte, mal cell), Epidermal basal cell (stem cell), Keratinocyte of Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, fingernails and toenails, Nail bed basal cell (stem cell), Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Medullary hair shaft cell, Cortical hair shaft cell, Cuticular Stem cells and committed progenitors for the blood and hair shaft cell, Cuticular hair root sheath cell, Hair root immune system (various types), Pluripotent stem cells, sheath cell of Huxley's layer, Hair root sheath cell of Totipotent stem cells, Induced pluripotent stem cells, adult Henle’s layer, External hair root sheath cell, Hair matrix cell stem cells, Sensory transducer cells, Autonomic neuron (stem cell), Wet stratified barrier epithelial cells, Surface cells, Sense organ and peripheral neuron Supporting cells, epithelial cell of Stratified squamous epithelium of cornea, Central nervous system neurons and glial cells, Lens cells, tongue, oral cavity, esophagus, anal canal, distal urethra and Pigment cells, Melanocyte, Retinal pigmented epithelial vagina, basal cell (stem cell) of epithelia of cornea, tongue, cell, Germ cells, Oogonium/Oocyte, Spermatid, Spermato oral cavity, esophagus, anal canal, distal urethra and vagina, cyte, Spermatogonium cell (stem cell for spermatocyte), Urinary epithelium cell (lining urinary bladder and urinary Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell ducts), Exocrine secretory epithelial cells, Salivary gland (in testis), Thymus epithelial cell, Interstitial cells, and mucous cell (polysaccharide-rich secretion), Salivary gland Interstitial kidney cells. serous cell (glycoprotein enzyme -rich secretion), Von 0263. A subject system introduced into a cell can be used Ebner's gland cell in tongue (washes taste buds), Mammary for regulating expression of a target polynucleotide (e.g., gland cell (milk secretion), Lacrimal gland cell (tear secre gene expression). In an aspect, the disclosure provides tion), Ceruminous gland cell in ear (wax secretion), Eccrine methods of regulating expression of a target polynucleotide Sweat gland dark cell (glycoprotein secretion), Eccrine in a cell. In some embodiments, the method comprises (a) Sweat gland clear cell (Small molecule secretion). Apocrine exposing a chimeric receptor polypeptide to an antigen, US 2017/O 1983O8 A1 Jul. 13, 2017

wherein (i) the chimeric receptor polypeptide is modified least 6 hours, at least 7 hours, at least 8 hours, at least 12 upon exposure to the antigen, and (ii) receptor modification hours, at least 16 hours, at least 20 hours, at least 24 hours, comprises a conformational change or a chemical modifi at least 2 days, at least 3 days, at least 4 days, at least 5 days, cation; (b) binding a chimeric adaptor polypeptide to the at least 6 days, at least 1 week, at least 2 weeks, at least 3 receptor in response to the modification to form a complex weeks, at least 1 month or longer. between a gene modulating polypeptide (GMP) and a cleav 0267 A chimeric receptor polypeptide can bind any age moiety, wherein the GMP comprises an actuator moiety Suitable antigen as described herein. In some embodiments, linked to a cleavage recognition site; and (c) cleaving the the chimeric receptor polypeptide is a transmembrane recep cleavage recognition site with the cleavage moiety, wherein tor. In some embodiments, the chimeric receptor polypeptide upon cleavage of the cleavage recognition site, the actuator is an intracellular receptor. In some embodiments, the chi moiety is activated to complex with a target polynucleotide. meric receptor polypeptide is a nuclear receptor. The antigen In some embodiments, the GMP forms a portion of an interacting domain of a chimeric receptor polypeptide can intracellular region of the chimeric receptor polypeptide, bind a membrane bound antigen, for example an antigen and the cleavage moiety forms part of the chimeric adaptor bound to the extracellular Surface of a cell (e.g., a target polypeptide. In some embodiments, the GMP forms a por cell). In some embodiments, the antigen interacting domain tion of the chimeric adaptor polypeptide, and the cleavage binds a non-membrane bound antigen, for example an moiety forms a portion of an intracellular portion of the extracellular antigen that is secreted by a cell (e.g., a target chimeric receptor polypeptide. In some embodiments, the cell) or an antigen located in the cytoplasm of a cell (e.g., a cleavage moiety is complexed with a second adaptor poly target cell). Antigens (e.g., membrane bound and non peptide that binds the receptor in response to the receptor membrane bound) can be associated with a disease such as modification, and the GMP forms a portion of the chimeric a viral, bacterial, and/or parasitic infection; inflammatory adaptor polypeptide. and/or autoimmune disease; or neoplasm such as a cancer 0264. A chimeric receptor polypeptide can be any chi and/or tumor. meric receptor polypeptide described herein. In some 0268. Upon exposure to the antigen, the chimeric recep embodiments, the chimeric receptor polypeptide is a trans tor can undergo receptor modification. Receptor modifica membrane receptor. For example, a chimeric transmem tion can comprise a conformational change, chemical modi brane receptor polypeptide comprises a G-protein coupled fication, or combination thereof. A chemical modification receptor (GPCR) such as Wnt receptor (e.g., Frizzled family can comprise, for example, phosphorylation or dephospho receptors); integrin receptor, cadherin receptor, catalytic rylation of at least one amino acid residue of the receptor. receptor including receptors possessing enzymatic activity Phosphorylation and/or dephosphorylation can occurat, for and receptors which, rather than possessing intrinsic enzy example, a tyrosine, serine, threonine, or any other Suitable matic activity, act by Stimulating non-covalently associated amino acid residue of a chimeric receptor polypeptide. enzymes (e.g., kinases); death receptor Such as members of Binding of a chimeric adaptor polypeptide to the chimeric the tumor necrosis factor receptor Superfamily; immune receptor polypeptide in response to receptor modification receptor Such as T-cell receptors; or any derivative, variant, can form a complex between a GMP and a cleavage moiety. or fragment thereof. In some embodiments, the receptor Formation of a complex between the GMP and cleavage does not comprise SEQ ID NO:39. moiety can result in cleavage of the cleavage recognition site 0265 Exposing a chimeric receptor polypeptide by the cleavage moiety. In some embodiments, the cleavage expressed in a cell to an antigen can be conducted in vitro recognition site comprises a polypeptide sequence (e.g., a and/or in vivo. Exposing a chimeric receptor polypeptide peptide cleavage domain) recognized by a cleavage moiety expressed in a cell to an antigen can comprise to bringing the comprising protease activity. The cleavage moiety can com receptor in contact with the antigen, which can be a mem prise protease activity which recognizes the polypeptide brane-bound antigen or non-membrane bound antigen. The sequence. A cleavage moiety comprising protease activity antigen is, in Some cases, bound the membrane of a cell. The can be a protease including, but not limited, to any protease antigen is, in some cases, not bound the membrane of a cell. described elsewhere herein, or any derivative, variant or Exposing a cell to an antigen can be conducted in vitro by fragment thereof In some embodiments, the cleavage rec culturing the cell expressing a subject system in the presence ognition site comprises multiple cleavage recognition of the antigen. For example, a cell expressing Subject system sequences, and each cleavage recognition sequence can be can be cultured as an adherent cell or in Suspension, and the recognized by the same or different cleavage moiety com antigen can be added to the cell culture media. In some prising protease activity (e.g., protease). In some embodi cases, the antigen is expressed by a target cell, and exposing ments, receptor modification comprises modification at mul can comprise co-culturing the cell expressing a subject tiple modification sites, and each modification is effective to system and the target cell expressing the antigen. Cells can bind a chimeric adaptor polypeptide. In some embodiments, be co-cultured in various suitable types of cell culture media, (i) the GMP forms a portion of the chimeric adaptor poly for example with Supplements, growth factors, ions, etc. peptide, (ii) the chimeric adaptor polypeptide is released Exposing a cell expressing a subject system to a target cell from the chimeric receptor polypeptide following cleavage (e.g., a target cell expressing an antigen) can be accom of the cleavage recognition site, and (iii) a further chimeric plished in vivo, in some cases, by administering the cells to adaptor polypeptide comprising a GMP binds the modified a subject, for example a human Subject, and allowing the receptor. cells to localize to the target cell via the circulatory system. 0269. In some embodiments, the cleavage recognition 0266 Exposing can be performed for any suitable length site comprises a first portion of an intein sequence that reacts of time, for example at least 1 minute, at least 5 minutes, at with the second portion of the intein sequence to release the least 10 minutes, at least 30 minutes, at least 1 hour, at least actuator moiety. A heterologous split intein system, as 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at described elsewhere herein, can be used to facilitate release US 2017/O 1983O8 A1 Jul. 13, 2017 42 of the actuator moiety. The actuator moiety can be cova wild-type nuclease, a variant thereof, a derivative thereof, or lently linked to the first portion of the intein sequence. The a fragment thereof as described elsewhere herein. The actuator moiety can be linked via its N-terminus or C-ter actuator moiety can regulate expression and/or activity of a minus to the first portion of the intein sequence. The gene or edit the sequence of a nucleic acid (e.g., a gene cleavage moiety can comprise the second portion of the and/or gene product). In some embodiments, the actuator intein sequence. The first portion or second portion of the moiety comprises a DNA nuclease Such as an engineered intein sequence can be the N-terminal intein, the C-terminal (e.g., programmable or targetable) DNA nuclease to induce intein, or any other Suitable portion of an intein that can genome editing of a target DNA sequence. In some embodi facilitate release of the actuator moiety. The intein sequences ments, the actuator moiety comprises a RNA nuclease Such can be from any Suitable source. The first and second portion as an engineered (e.g., programmable or targetable) RNA can be from the same or different sources (e.g., organism, nuclease to induce editing of a target RNA sequence. In protein). In an illustrative example, an actuator moiety can Some embodiments, the actuator moiety has reduced or be covalently linked (e.g., at its N-terminus or C-terminus) minimal nuclease activity. An actuator moiety having via a peptide bond to a first portion of the intein sequence, reduced or minimal nuclease activity can regulate expres which comprises an N-terminal intein. The actuator moiety sion and/or activity by physical obstruction of a target N-terminal intein fusion can be contacted with a second polynucleotide or recruitment of additional factors effective portion of the intein sequence comprising a C-terminal to Suppress or enhance expression of the target polynucle intein. This contacting of the first and second portion of the otide. In some embodiments, the actuator moiety comprises intein sequences can result in a site specific cleavage (e.g., a nuclease-null DNA binding protein derived from a DNA at a site between the actuator moiety and the N-terminal nuclease that can induce transcriptional activation or repres intein), thereby releasing the actuator moiety. In another sion of a target DNA sequence. In some embodiments, the illustrative example, an actuator moiety can be covalently actuator moiety comprises a nuclease-null RNA binding linked (e.g., at its N-terminus or C-terminus) via a peptide protein derived from a RNA nuclease that can induce bond to a first portion of the intein comprising a C-terminal transcriptional activation or repression of a target RNA intein. The actuator moiety-C-terminal intein fusion can be sequence. In some embodiments, the actuator moiety is a contacted with a second portion of the intein sequence nucleic acid-guided actuator moiety. In some embodiments, comprising an N-terminal intein. This contacting of the first the actuator moiety is a DNA-guided actuator moiety. In and second portion of the inteins can result in a site-specific Some embodiments, the actuator moiety is an RNA-guided cleavage (e.g., at a suitable site between the actuator moiety actuator moiety. An actuator moiety can regulate expression and the C-terminal intein), thereby releasing the actuator or activity of a gene and/or edit a nucleic acid sequence, moiety. whether exogenous or endogenous. Upon cleavage of the 0270. In some embodiments, the cleavage recognition cleavage recognition site, the actuator moiety is activated to site comprises a disulfide bond. In some embodiments, the complex with a target polynucleotide. cleavage moiety comprises oxidoreductase activity. The 0272 Any suitable nuclease can be used in an actuator disulfide bond can link the actuator moiety to a portion of the moiety. Suitable nucleases include, but are not limited to, chimeric adaptor polypeptide or chimeric receptor polypep CRISPR-associated (Cas) proteins or Cas nucleases includ tide. The disulfide bond can be formed by one or more ing type I CRISPR-associated (Cas) polypeptides, type II cysteines of the actuator moiety. The cysteines can be CRISPR-associated (Cas) polypeptides, type III CRISPR engineered into the actuator moiety. The cysteines can be a associated (Cas) polypeptides, type IV CRISPR-associated part of the native or wild-type sequence of the actuator (Cas) polypeptides, type V CRISPR-associated (Cas) poly moiety. The cysteines can be present in a linker peptide peptides, and type VI CRISPR-associated (Cas) polypep appended to the actuator moiety. Cleavage of the disulfide tides; Zinc finger nucleases (ZFN); transcription activator bond can be facilitated by, for example, altering the redox like effector nucleases (TALEN); meganucleases; RNA conditions of the disulfide bond. Alteration of the redox binding proteins (RBP); CRISPR-associated RNA binding conditions can lead to reduction of the disulfide bond to proteins; recombinases; flippases; transposases; Argonaute thiols and release of the actuator moiety. Cleavage of the proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argo disulfide bond can be facilitated by a cleavage moiety naute (aAgo), and eukaryotic Argonaute (eAgo)); any comprising a redox agent that can lead to reduction of the derivative thereof any variant thereof and any fragment disulfide bond. The redox agent can be an enzyme, or any thereof. derivative, variant or fragment thereof. The enzyme can be 0273. In some embodiments, the actuator moiety com an oxidoreductase. Examples of oxidoreductases include prises a Cas protein that forms a complex with a guide protein-disulfide reductase, thioredoxins, glutaredoxins, nucleic acid, such as a guide RNA. In some embodiments, thiol disulfide oxidoreductases (e.g., DsbA, BdbA-D, the actuator moiety comprises a RNA-binding protein (RBP) MdbA, SdbA), and glutathione disulfide reductase. The optionally complexed with a guide nucleic acid, such as a redox agent can be from any suitable source including guide RNA, which is able to form a complex with a Cas prokaryotes and eukaryotes. Cofactors (e.g., nicotinamide protein. In some embodiments, the actuator moiety com cofactors, flavins, and derivatives and analogs thereof) can prises a Cas protein lacking cleavage activity. be supplied for optimal activity of the enzyme. 0274 The actuator moiety of a subject system can bind to 0271. A GMP, as described elsewhere herein, can com a target polynucleotide to regulate expression and/or activity prise an actuator moiety linked to a cleavage recognition of the target polynucleotide by physical obstruction of the site. The actuator moiety can comprise a nuclease (e.g., target polynucleotide or recruitment of additional factors DNA nuclease and/or RNA nuclease), modified nuclease effective to Suppress or enhance expression of the target (e.g., DNA nuclease and/or RNA nuclease) that is nuclease polynucleotide. In some embodiments, the actuator moiety deficient or has reduced nuclease activity compared to a comprises a transcriptional activator effective to increase US 2017/O 1983O8 A1 Jul. 13, 2017 43 expression of the target polynucleotide. The actuator moiety nucleotides. Examples of target polynucleotides include a can comprise a transcriptional repressor effective to decrease sequence associated with a signaling biochemical pathway, expression of the target polynucleotide. e.g., a signaling biochemical pathway-associated gene or 0275. In some embodiments, the target polynucleotide polynucleotide. Examples of target polynucleotides include comprises genomic DNA. In some embodiments, the target a disease associated gene or polynucleotide. A "disease polynucleotide comprises a region of a plasmid, for example associated gene or polynucleotide refers to any gene or a plasmid carrying an exogenous gene. In some embodi polynucleotide which is yielding transcription or translation ments, the target polynucleotide comprises RNA, for products at an abnormal level or in an abnormal form in cells example mRNA. In some embodiments, the target poly derived from a disease-affected tissue compared with tissue nucleotide comprises an endogenous gene or gene product. (s) or cells of a non-disease control. In some embodiments, The actuator moiety can include one or more copies of a it is a gene that becomes expressed at an abnormally high nuclear localization signal that allows the actuator to trans level. In some embodiments, it is a gene that becomes locate into the nucleus upon cleavage from the GMP expressed at an abnormally low level. The altered expression 0276 A target polynucleotide of the various embodi can correlate with the occurrence and/or progression of the ments of the aspects herein can be DNA or RNA (e.g., disease. A disease-associated gene also refers to a gene mRNA). The target polynucleotide can be single-stranded or possessing mutation(s) or genetic variation that is directly double-stranded. The target polynucleotide can be genomic responsible or is in linkage disequilibrium with a gene(s) DNA. The target polynucleotide can be any polynucleotide that is response for the etiology of a disease. The transcribed endogenous or exogenous to a cell. For example, the target or translated products may be known or unknown, and may polynucleotide can by a polynucleotide residing in the be at a normal or abnormal level. nucleus of a eukaryotic cell. The target polynucleotide can 0278 Examples of disease-associated genes and poly be a sequence coding a gene product (e.g., a protein) or a nucleotides are available from McKusick-Nathans Institute non-coding sequence (e.g., a regulatory polynucleotide). In of Genetic Medicine, Johns Hopkins University (Baltimore, Some embodiments, the target polynucleotide comprises a Md.) and National Center for Biotechnology Information, region of a plasmid, for example a plasmid carrying an National Library of Medicine (Bethesda, Md.), available on exogenous gene. In some embodiments, the target poly the World Wide Web. Exemplary genes associated with nucleotide comprises RNA, for example mRNA. In some certain diseases and disorders are provided in Tables 3 and embodiments, the target polynucleotide comprises an 4. Examples of signaling biochemical pathway-associated endogenous gene or gene product. genes and polynucleotides are listed in Table 5. 0277. The target polynucleotide may include a number of 0279 Mutations in these genes and pathways can result disease-associated genes and polynucleotides as well as in production of improper proteins or proteins in improper signaling biochemical pathway-associated genes and poly amounts which affect function. TABLE 3 DISEASE DISORDERS GENE(S) Neoplasia PTEN: ATM; ATR: EGFR; ERBB2; ERBB3; ERBB4; Notch1: Notch2: Notch3: Notch4; AKT: AKT2: AKT3; HIF: HIF1a; HIF3a; Met; HRG; Bcl2: PPAR alpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a: APC: RB (retinoblastoma); MEN1; VHL. BRCA1: BRCA2; AR (Androgen Receptor); TSG 101; IGF: IGF Receptor; Igfl (4 variants); Igf2 (3 variants); Igf1 Receptor; Igf2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Cel2; Ce2; cp (ceruloplasmin); Timp3; cathepsin D; Degeneration Vldlr; Cer2 Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4); COMT, DRD (Drd1a); SLC6A3; DAOA: DTNBP1; Dao (Dao1) Trinucleotide Repeat HTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Disorders DX); FXNX25 (Friedrich's Ataxia): ATX3 (Machado oseph's DX): ATXN1 and ATXN2 (spinocerebellar ataxias): DMPK (myotonic dystrophy); Atrophin-1 and Atin1 (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR (Alzheimer's); AtXn7: AtXn 10 Fragile X Syndrome FMR2; FXR1; FXR2: mGLUR5 Secretase Related Disorders APH-1 (alpha and beta); Presenilin (Psen1); nicastrin (Ncstn); PEN-2 Others Nos.1; Parp1; Natl: Nat2 Prion - related disorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a: VEGF-b; VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol): GRIA2; Grms: Grin1; Htr1b; Grin2a: Drd3: Pdyn; Grial (alcohol)

US 2017/O 1983O8 A1 Jul. 13, 2017 46

TABLE 5-continued

CELLULARFUNCTION GENES

ERK/MAPK Signaling

Glucocorticoid Receptor Signaling

Axonal Guidance Signaling

Ephrin Receptor Signaling

Actin Cytoskeleton Signaling

Huntington's Disease Signaling

Apoptosis Signaling US 2017/O 1983O8 A1 Jul. 13, 2017 47

TABLE 5-continued

CELLULARFUNCTION GENES

S. Integrin Signaling

Xenobiotic Metabolism Signaling

SAPK/JNK Signaling

US 2017/O 1983O8 A1 Jul. 13, 2017 49

TABLE 5-continued

CELLULARFUNCTION GENES Fc Epsilon RI Signaling

G-Protein Coupled Receptor Signaling

Inositol Phosphate Metabolism

PDGF Signaling

VEGF Signaling

Natural Killer Cell Signaling

Cell Cycle: G1/S Checkpoint Regulation

T Cell Receptor Signaling

Death Receptor Signaling

FGF Signaling

GM-CSF Signaling

Amyotrophic Lateral Sclerosis Signaling

JAK/Stat Signaling

Nicotinate and Nicotinamide Metabolism

Chemokine Signaling US 2017/O 1983O8 A1 Jul. 13, 2017 50

TABLE 5-continued

CELLULARFUNCTION GENES IL-2 Signaling

Synaptic Long Term Depression

Estrogen Receptor Signaling

Protein Ubiquitination Pathway

IL-10 Signaling

WDRRXRActivation

TGF-beta Signaling E s Toll-like Receptor Signaling

R N p38 MAPK Signaling H C

Neurotrophin TRK Signaling

FXRRXR Activation

Synaptic Long Term Potentiation

Calcium Signaling

EGF Signaling

Hypoxia Signaling in the Cardiovascular System LPS, IL-1 Mediated Inhibition of RXR Function

LXRRXR Activation

Amyloid Processing

IL-4 Signaling

Cell Cycle: G2/M DNA Damage Checkpoint Regulation Nitric Oxide Signaling in the Cardiovascular System