(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/094874 Al 16 June 2016 (16.06.2016) W P O P C T (51) International Patent Classification: (74) Agents: KOWALSKI, Thomas J. et al; Vedder Price C12N 15/11 (2006.01) P.C., 1633 Broadway, New York, NY 1001 9 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US2015/065396 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) International Filing Date: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 11 December 2015 ( 11.12.2015) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 62/091,456 12 December 2014 (12. 12.2014) US SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 62/180,692 17 June 2015 (17.06.2015) US TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: THE BROAD INSTITUTE INC. [US/US]; (84) Designated States (unless otherwise indicated, for every 415 Main Street, Cambridge, MA 02142 (US). MAS¬ kind of regional protection available): ARIPO (BW, GH, SACHUSETTS INSTITUTE OF TECHNOLOGY GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, [US/US]; 77 Massachusetts Ave., Cambridge, MA 02142 TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (US). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: DAHLMAN, James, E.; 86 Rice Street, Cam LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, bridge, MA 02140 (US). ZHANG, Feng; 100 Pacific SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Street, Apt. 11, Cambridge, MA 02139 (US). GW, KM, ML, MR, NE, SN, TD, TG). [Continued on nextpage] (54) Title: ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS (57) Abstract: The present invention generally relates to CR- ISPR systems or complexes, such as those with an escorted Cell-penetrating aptamer 1 (MS2 Loop) g u ide RNA. Cell-penetrating aptamer 2 {Stem Loop 2) This aptamer would be called; C1 Otter Γ ΤΤ Τ c Yi "" ' r M n 3 " UGCGAA ¾ υυ c Mjn ACGCUU CAA GGAGU - , cGUGC^ m„ ... CCUCAS CACG & 00 FIG. 1 © v o o w o 2016/094874 A llll II II 11III III II 11 III I II II III II I II Published: before the expiration of the time limit for amending the — with international search report (Art. 21(3)) claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS RELATED APPLICATIONS AND INCORPORATION BY REFERENCE [0001] This application claims priority from US application Serial No. 62/091,456, filed December 12, 2014, and US application Serial No. 62/180,692, filed June 17, 2015. [0002] The foregoing application(s) and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [0003] Mention is made of LIS applications 62/091,455, filed December 12, 2014, 62/096,708, filed December 24, 2014, 62/180,709, filed June 17, 201 5, and PCT/US201 5/65385 (Broad Institute reference no. BI-2014/100.WO1, attorney docket 47627.99.2001) entitled PROTECTED GUIDE RNAS (PGRNAS). Mention is also made of US applications 62/091,462, filed December 12, 2014, 62/096,324, filed December 23, 2014, 62/180,681, filed June 17, 2015 62/237,496, filed October 5, 2015, and PCT/US20 15/65393 entitled DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS. FEDERAL- FUNDING LEGEND [0004] This invention was made with government support under grant numbers MH1 00706 and MH1 10049 awarded by the National Institutes of Health The government has certain rights in the invention. FIELD OF THE INVENTION [0005] The present invention generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof!, including modified guides for CRISPR systems or complexes. Thus, the present invention generally relates to methods and compositions used for the spatial and temporal control of gene expression, such as genome perturbation BACKGROUND OF THE INVENTION [0006] Normal gene expression is a dynamic process with carefully orchestrated temporal and spatial components, the precision of which are necessary for normal development, homeostasis, and advancement of the organism. I turn, the d singulation of required gene expression patterns, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. [0007] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE IN VENTION [0008] In one aspect the invention provides escorted CRISPR-Cas systems or complexes, especially such a system involving an escorted CRISPR-Cas system guide. It will be appreciated that reference herein to the Cas protein is restricted to Cas9, including SpCas9, SaCas9 and other orthologs. [0009] By "escorted" is meant that the CRISPR-Cas system or complex or guide is delivered to a selected time or place within a cell, so that activity of the CRISPR-Cas system or complex or guide is spatially or temporally controlled. For example, the activity and destination of the CRISPR-Cas system or complex or guide may be controlled by an escort RNA aptamer sequence that has binding affinity for an aptamer ligand, such as a ce l surface protein or other localized cellular component. Alternatively, the escort aptamer (escort RNA aptamer sequence) may for example be responsive to, i.e. activated or inactivated by, an aptamer effector on or in the cell. The aptamer effector may be a transient effector, such as an external energy source that is applied to the cell at a particular time. In some embodiments, the external energy source is light energy. [0010] It will be appreciated that the terms "escort RNA aptamer sequence" and "escort aptamer" are used interchangeably herein. In some embodiments, the escort RNA aptamer sequence comprises an aptamer sequence and is fused to the guide at one or more of the tetraloop and/or stem loop 2 . Preferably, the escort RNA aptamer sequence is completely, RNA although it may comprise DNA or other nucleotides: preferably it is predominantly, i.e. at least 50%, RNA. Examples of aptamers comprised within the escort RNA aptamer sequence include Otter and CI, as well as their minimal versions: OtterMin and CIMin. [00 ] The invention involves sgRNA of CRISPR-Cas systems or complexes and hence such complexes or systems having an sgRNA with a functional structure designed to improve sgRNA structure, architecture, stability, genetic expression, or any combination thereof. Such a structure can include an aptamer. Accordingly, the invention provides an sgRNA modified, e.g., by one or more aptamer(s) designed to improve sgRNA delivery, including delivery across the cellular membrane, to intracellular compartments, or into the nucleus. Such a structure can include, either in addition to the one or more aptamer(s) or without such one or more aptamer(s), moiety(ies) so as to render the guide deliverable, inducible or responsive to (for example activatable or inactivatable by) a selected effector. The invention accordingly comprehends an sgRNA that responds to normal or pathological physiological conditions, including without limitation pH, hypoxia, 0 2 concentration, temperature, protein concentration, enzymatic concentration, lipid structure, light exposure, mechanical disruption (e.g. ultrasound waves), magnetic fields, electric fields, or electromagnetic radiation. [0012] The modified sgRNA with a functional staicture can be administered through a wide variety of means, including intravenous, oral, intramuscular, subcutaneous, topical, intraocular, intracerebroventricular, sublingual, rectal, vaginal, intrathecal, intraperitoneal, intradermal, transdermal, or intraspinal routes, by inhalation, vaporization, or nebulization, or by direct access to tissue following surgical exposure of the pancreas, kidney, peritoneal cavity, intestine, stomach, heart, lungs, liver, spleen, nerves, brain, or spinal cord. [0013] An aspect of the invention provides non-natural ly occurring or engineered composition comprising an escorted single guide RNA (esgRNA) comprising: an RNA guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a ceil; and, an escort RNA aptamer sequence, wherein the escort aptamer (i.e. the escort RNA aptamer sequence) has binding affinity for an aptamer iigand on or in the cell, or the escort aptamer (i.e. the escort RNA aptamer sequence) is responsive to a localized aptamer effector on or in the cell, wherein the presence of the aptamer ligand or effector on or in the cell is spatially or temporally restri cted [0014] In some embodiments, the escort RNA aptamer sequence has binding affinity for an aptamer ligand on or in the cell.