Efficient Delivery of Nuclease Proteins for Genome Editing in Human Stem

© 2015 Nature America, Inc. All rights reserved. bases, with occasional target site overlap from an adjacent zinc- adjacent an from overlap site target occasional with bases, the within situated recognition DNA mediate that acids amino the a in residues acid amino ~30 of consist domains zinc-finger Individual cleavage. DNA for required are monomers TALEN and ZFN two dimer, a as functions domain cleavage FokI the Because infection. promote that factors lence viru host regulate in they which bacteria, pathogenic from plant derived are TALEproteins whereas genome, human the in teins pro occurring frequently most the among are zinc-fingers lar, and TAL effector DNA-binding domains, respectively. In particu of the FokI restriction endonuclease and customizable zinc-finger (RGENs)). endonucleases RNA-guided as known (also Cas9 used to facilitate genome editing are ZFNs, TALENs and CRISPR/ commonly most are that technologies three The diseases. many of causes genetic underlying the to correct a means by providing medicine transform to potential the has it and research, logical has for customized basic revolutionized bio genome engineering homology-directed via (HDR) (NHEJ) repair correction or joining integration end targeted (ii) nonhomologous error-prone by knockout gene (i) outcomes: two of one to leads typically which breaks (DSBs) that stimulate the cellular DNA repair double-strand machinery DNA targeted induce to configured are systems sequence genomic any organisms and model types cell a of across range broad virtually manipulating of that capable tools are flexible highly are endonucleases DNA Site-specific I expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 ared. described, along with procedures for evaluating nuclease protein activity. moieties; and which are intrinsically cell-permeable; editing in human embryonic stem cells and primary cells. particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based togenome fewer off-target effects while maintaining high rates of targeted modification. on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action ofand thesefast genome-modifyingturnover, technologies leadingrequires their safe and efficient delivery into relevant cell types. with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, clusteredrealizing regularly the fullinterspaced potential short palindromic repeat ( T Published online 22 October 2015; contributed equally to this work. University, Seoul, South Korea. and Biology of Fruit Crop, Research and Innovation Centre, Fondazione Edmund Mach (FEM), San Michele all’Adige, Italy.The Scripps Research Institute, La Jolla, California, USA. Institute, La Jolla, California, USA. 1 Kanchiswamy Jia Liu editing in human stem cells and primary cells Efficient delivery of nuclease proteins for genome 1842 proteins zinc-finger Synthetic domain. finger Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China. argeted nucleases, including zinc-finger nucleases (ZF NTRO protocol ZFNs α

| -helical domain -helical VOL.10 NO.11VOL.10 D 1 1 UCT 0 , 9 and TALENs , Thomas Gaj I ON 7– C as9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, 9 6 . The ease with which these technologies can be be can technologies these which with ease .The , Jin-Soo Kim | 2015 1 1 2 1 . Each zinc-finger typically binds three three binds typically zinc-finger Each . 8 are fusions between the cleavage domain Present address: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA. | 10 2 natureprotocols – 3 Deceased. Correspondence should be addressed to J.L. ( Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA. 4 doi:10.1038/nprot.2015.11 , 8 , 9 , Yifeng Yang 5 , 7 & Carlos F III Barbas TALEN β β α 5 Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. configuration, with with configuration, proteins, which can be internalized via conjugation with cell-penetrating peptide 1 3 1 can be generated generated be can , Nan Wang 7 N 1– s), transcription activator-like ( 3 CR P . These rotocols for the expression, purification and delivery of ZF 5, I 6 SPR 2 1 or or – , Sailan Shui 4 4 - - - - , , )/ 10

CR box capable of achieving sequence-specific tool genomic modification. powerful a provide technologies foundational three these elements tracrRNA and crRNA the both contains that (sgRNA) RNA guide single a and engineering site target the of downstream sequence motif adjacent protospacer conserved a and crRNA the within sequence 20-nt a on nucle primarily Cas9 relies the ase recognition, target For proteins. Cas by DNA pathogenic of cleavage sequence-specific direct that (tracrRNA) invariant and (crRNA) RNA CRISPR protein short Cas9 sequence-variable of the expression through by achieved is This cleavage DNA RNA-guided foreign via invading DNA against immunity acquired provides system CRISPR the bacteria, from derived Originally design. CRISPR/Cas9 minimal the assembly, their with editing RNA-guided genome efficient highly enables system for methods engineering recognizing DNA sequence of contiguous any nearly capable arrays TALE the synthetic of of construction Elucidation code (RVDs). recognition DNA protein effector di-residues TAL variable termed are repeat which domain, the each within present residues acid amino hypervariable two by determined is repeat effector TAL each of DNA.of pair base a The single specificity each recognizes that repeats 35-aa to 33- of series a of composed is that domain prede ( with specificity fined each domains, zinc-finger preselected of library a using by or selection by sequences of range wide a recognize to I SPR [email protected] In contrast to ZFNs and TALENs, which rely on protein protein on rely which TALENs, and ZFNs to contrast In -associated protein 9 ( 1 O , Sojung Kim nce they are constructed, nuclease proteins can be 2 18–21 The Skaggs Institute for Chemical Biology, The Scripps Research T hese features make nuclease protein delivery 1 , and it now consists of only the Cas9 nuclease nuclease Cas9 the only of consists now it and Box Box 7 . This system has been simplified for genome genome for simplified been has system This . TAL 1 ) or J.-S.K. ( ) effector nucleases ( ). TALE proteins contain a DNA-binding DNA-binding a contain ). TALE proteins 4 5 Department of Cell and Molecular Biology, 1 , Chidananda Nagamangala 7 Tgte, ih Fs n TALENs, and ZFNs with Together, . 7 C Department of Chemistry, Seoul National as9), have provided researchers [email protected] 6 U 1 Department of Genomics 6 nlike methods that rely ( trans- Box Box TALEN 14,1 1 ). activating crRNA crRNA activating ). N 5 proteins, has facilitated facilitated has s) and 9 These authors

1 7 - - - .

© 2015 Nature America, Inc. All rights reserved. Streptococcus pyogenes Streptococcus monomers, TALEN two of delivery the for a of AAVparticle use single the car prevents or hinders limited which kb), their (<5 capacity by rying constrained are however, vectors, AAV engineering genome nuclease-mediated facilitated resulting in efficient of of ZFNs and CRISPR/Cas9 into both mouse liver (AAV)virus adeno-associated vectors have the facilitated delivery types cell broad primary a of into range nucleases delivering for means a provide vectors RNA. labile handling with associated challenges and burdens additional the it imposes and accompany technologies, that transfection constraints chemical and the electroporation of many to subject remains approach this backbone, DNA the of insertion chromosomal of risk the alleviating for route a offers mRNA nuclease-encoding of transfection Although genome. host the into DNA foreign of mutagenesis insertional for potential the notably, and, escence) qui exhibit or slowly divide that cells primary for (particularly reagents transfection chemical or process toxicity practicality, from including the electroporation and effectiveness their limit that drawbacks with associated are techniques these of all However, HDR. for DNA donor of ence straightforward and cost-effective approach for nucleases introducing into cells alone or in a the pres provide methods These phosphate nucleofection electroporation as such techniques, standard of number a by cells into delivered be can DNA plasmid Nuclease-encoding Methods for delivering nucleases into cells applications and therapeutic research for basic types cell into various delivery their safe that and facilitate methods the of efficient development requires platforms these of potential full the However,realizing sequences canbecustom-synthesized and cloned directly into anumber of previously described CRISPR plasmid backbones protospacer adjacent motif (PAM) recognized by SpCas9. Sense and antisense oligonucleotides encoding the selected sgRNA protospacer the SpCas9protein, search the selectedgene sequence for the motif 5 starting bysearching for potential Cas9cleavage sitesusing anonline CRISPR design toolorDNAsequencing viewing software. For for genome-scale applications solid-phase assembly the repetitive nature of the TAL effectorrepeats poseproblems for their amplification. High-throughput approaches basedon approach for assembling these isthe Golden Gatecloning method have alsobeendescribed consider context-dependent effects Selection-based procedures, including oligomerized poolengineering (OPEN),canfacilitate isolation of zinc-finger arrays that generated byselection orrational design. Naturally occurring zinc-finger domains canalsobeincorporated into this method which isbasedonthe useof apreselected library of zinc-finger modules thatare capable of recognizing individual DNAtriplets have beendescribed elsewhere, butthey are reviewed below. nuclease platform used, avariety of approaches canbeimplemented for their assembly. Detailedprotocols for many of these methods Before nuclease proteins canbeintroduced into cells, the genes encoding these proteins must beconstructed. Depending onthe In contrast to electroporation or chemical transfection, viral viral transfection, chemical or electroporation to contrast In Box 1 Unlike zinc-fingers and TALEs, no protein engineering is necessary for redirecting the CRISPR system to new target sites. We recommend Numerous methods havealsobeendeveloped thatenable assemblyof synthetic TAL effectorarrays. Perhaps the simplest Synthetic zinc-fingers withuniqueDNA-binding specificitycanbe readilyconstructed using the ‘modular assembly’approach 2 6 22,2 | , cationic liposomes cationic , Methodsforconstructingtargeted nucleases 2 5 3 , as well as chemical methods, including calcium calcium including methods, chemical as well ,as . in in vivo 9 Cas9 (SpCas9) with its sgRNA its with (SpCas9) Cas9 9 and ligation-independent cloning techniques 94,9 genome editing. AAV vectors have also 5 in vitro in , including those preselected for context-dependent interactions 50,99,10 2 7 3 , polymers , 9 2 3 , low transfection efficiency efficiency transfection low , 0 . Approaches for building extended arrays around two-orthree-finger zinc-finger building blocks and and . in vivo in 2 8 or peptides or 33–3 . In particular, particular, In . 5 in vitro in and brain 4 0 and, in in and, 37–3 2 29,3 4 or or 3 3 0 9 6 - - - 1 . , . 1

6 . PCR-basedmethods havealsobeendescribed 10 acids nucleic from cell the within nuclease the expressing than rather protein purified as cells into nucleases introducing by including present is nuclease cells the within that time of amount the limiting by is specificity nuclease improving for approach One applications. engineering genome myriad for consequences serious effects has that off-target increased to lead could DNA from time of for an period expression extended nuclease of levels domain DNA-binding effector TAL the within rearrangements to lead could baculovirus from genes TALEN of expression the although delivery, nuclease for systems vector baculoviral and HDR scarless mediating of capable are AdVs protein-capped in vivo nucleases for delivering means another provide (AdVs) vectors adenovirus in cells to that are recombination less susceptible arrays RVD tripartite preassembled variable, highly generate to used be can usage codon alternative and degeneracy acid amino domain DNA-binding this overcome the to help have developed strategies of the of nature because platforms repetitive vector both within truncations and rearrangements to prone are In genes effects. TALEN-encoding addition, silencing to prone are IDLVs and genome, within host the randomly integrates lentivirus However, technologies. screens functional genome-wide both editing genome vitro in facilitates also lentivirus Conventional DNA donor for HDR with cells into nucleases for delivering vectors (IDLVs),lentiviral in an provide contrast, means effective some cases, a a with nuclease donor template. Integrase-defective f eoi mdfcto. oitgaig gamma-retroviral Nonintegrating modification. rates genomic time- low of with and associated are they difficult and produce, to particularly consuming are however, vectors, These ′ 0 -GNNNNNNNNNNNNNNNNNNN-NGG-3 havealsobeenreported, enabling rapid assemblyof TALENs Regardless of the viral or nonviral vector system used, high high used, system vector nonviral or viral the of Regardless 6 2 53,5 . This approach offers the distinct advantage of rapid action 4 3 and and 51,5 4 . Unlike IDLVs,DNA,donor as exist free-ended which 61,6 2 icuig TALENs including , in vivo in 2 . This can be achieved in a number of ways of number a in achieved be can This . ● natureprotocols 4 4 96,9

, and it provides a means for performing performing for means a provides it and , T IMI 7 . N 6 0 57–5 . 45–4 G ~4–14d ′ 9 4 , where 5 7 have also been configured configured been also have 8 using CRISPR/Cas9-based CRISPR/Cas9-based using , into primary cells and and cells primary into ,

| VOL.10 NO.11VOL.10 9 8 ; however, ′ -NGG-3 49,5 protocol 5 6 0 1 . Alternatively, 18,10 0 , a drawback drawback a , 4

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© 2015 Nature America, Inc. All rights reserved. mary mary CD4 including types, cell primary pluripotent induced of variety wide a into delivered ( osmocytosis internalization CPP-mediated described RNP delivering for methods although addition, In lacking. are transgenes DNA containing donor with restrictions proteins nuclease specific delivering for more methods Specifically, of exist. also handful a approach, the limita to a tion represents variability broad this editing genome Although and efficiency. uptake of route levels variable to administration contribute and all platform nuclease type, cell Target nuclease protein of Limitations delivery procedures. these using achieved be can that ifications been recently described also have (ssODNs) oligonucleotides DNA single-stranded with and both engineering genomic for mediating delivery protein cells hair mouse and cells into using delivered nucleofection be can complex (RNP) ribonucleoprotein termini their to fused cally ( repeats TALeffector individual of surface tides (CPPs) that mediate cellular uptake can be conjugated to the lentivirus as using cells proteins into delivered also be can structure, repetitive and size their of vectors because viral several with which are incompatible TALENs, replacement. gene or integration targeted for cells into DNA donor of co-delivery for allows which of receptor-mediated last the transfer, using internalization endocytosis cellular direct for systems transduction protein conventional many exceed that rates at delivery protein proteins zinc-finger of ( activity cell-penetrating intrinsic the culture in cells direct to their after application knockout gene achieving of capable are and they cell-permeable, inherently are proteins ( ZFN particular, strategies In different of number a through protein fied three all date, To as puri cells into affect introduced have been nucleases of types major modification. adversely genome not targeted does of rates activity the of window shortened this Notably, effects. off-target fewer in resulting turnover, fast and introduced directly into cells via nucleofection or transfection of RNP. Cas9 proteinand is sulfurshown atomsin gray.colored red, sgRNA blueis andindicated yellow, by green respectively. sticks. ( Cys residues used for conjugation shown as spheres, with oxygen, nitrogen endowed via Cys-conjugation with cell-penetrating peptides. Surface-exposed PthXo1 TAL effector DNA-binding domain (PDB ID: the zinc-finger protein. ( penetrating activity of the ZFN protein because of the positive charge of to dark blue (positively charged residues; PDB ID: from dark red (negatively charged residues) to white (neutral residues) Cartoon of the electrostatic surface potential of a zinc-finger protein colored Figure 1 1844 tissue-specific i. 1 Fig. protocol We and others have shown that nuclease proteins can be be can proteins nuclease that shown have others and We in vivo in

| VOL.10 NO.11VOL.10 a

| ), which has recently been leveraged for intracellular intracellular for leveraged been recently has which ), Modes of nuclease protein delivery. ( 7 + 4 T cells . Strategies for combining nuclease protein delivery delivery protein nuclease combining for Strategies . , few approaches have been established that enable enable that established been have approaches few , 6 8 and retrovirus- and Fig. 1 Fig. in vivo in 62,6 | c 2015 72,7 6 b ) 4 4 delivery of nuclease proteins. nuclease of delivery in vivo in 7 66,6 ) Cell-penetrating TALEN protein. Cartoon of the , hematopoietic stem or progenitor cells and human embryonic stem cells and embryonic human 6 3 72–7 . , Lipofectamine-mediated transfection , Lipofectamine-mediated 7 | 6

. ZFN proteins can also be engineered engineered be also can proteins ZFN . 9 natureprotocols 4 . Alternatively, cell-penetrating pep cell-penetrating Alternatively, . 7 , further expanding the scope of mod 1 7 . Finally, a preformed Cas9-sgRNA Cas9-sgRNA preformed a Finally, . 4 5 , indicating the broad potential of of potential broad the indicating , 6 7 or lentivirus-mediated 62,64,6 5 and small-molecule–induced small-molecule–induced and 5 . This property is due to to due is property This . in vivo in a ) Cell-permeable ZFN protein. 2I1 3UG Fig. 1 Fig. 3 ) have recently been been recently have M 8 ) 5 8 . Intrinsic cell- 6 . Cell permeability b c ) Cas9 ribonucleoprotein. Cartoon of the Cas9 ribonucleoprotein (PDB ID: ) 7 0 or geneti or 6 72,7 9 protein protein Fig. Fig. ex ex vivo 5 , pri 64,6 1 7 ). 4 ------8 ,

of flexibility and efficiency: once recombinant Cas9 protein is is protein genomic loci user-specific only generated, requires modifying Cas9 the recombinant once efficiency: and degree flexibility highest of the our provides In RNP of application. target nucleofection the experience, for platform suitable most the types. cell of our have procedures to the potential be to applied a diverse range CD4 primary on Wefocus cells. into directly protein Cas9 and TALEN introducing ZFN, for protocols step-by-step three describe we Here design Experimental blood mononuclear cells (PBMCs) using ZFN proteins ZFN using (PBMCs) cells mononuclear blood CD4 primary in delivery. protein ZFN activity. measure accurately to order in samples ase-treated from nucle must cells be subtracted from cleavage mock-treated background assay, nuclease Surveyor the using editing genome no additives, Cas9 protein only or sgRNA only.containing When quantifying solution nucleofection with cells transfecting mend no or nucleases only one monomer.nuclease For RNP, we recom either containing medium serum-containing with cells treating For using ZFN experiments and TALEN proteins, we recommend treatments. mock including controls, relevant to perform crucial Controls. here. described procedures the for basis the activity maximum for protein of each For nuclease platform, we previously determined cells. the optimal amount into administration subsequent and expression over for vector expression bacterial pET-28 the into cloned are these After initial proof-of-principal experiments, nuclease-encoding lines. gene(s) cell human transformed in gene(s) encoding nuclease- of the activity evaluate to rapidly methods transfection transient described previously using Werecommend validated. (see sgRNA. new of production and gene editing activity editing gene and improves ZFN sequence protein uptake (NLS) signal localization nuclear (SV40) virus cells. simian the of in repeats tandem of poration mutagenesis incor of that demonstrated we levels methodology, this on high Toimprove for treatments protein consecutive required strategies delivery protein ZFN generation endocytosis cells primarily through macropinocytosis and caveolin-dependent enter domains DNA-binding zinc-finger that showed previously a Successful nuclease protein delivery first requires choosing choosing requires first delivery protein nuclease Successful Once a platform is selected, the nuclease must be constructed constructed be must nuclease the selected, is platform a Once Box Box 1 Finally, for any protein delivery experiment, it is is it experiment, delivery protein any for Finally, for an overview of nuclease assembly methods) and and methods) assembly nuclease of overview an for 6 6 + Dsie h smlct o ti tcnqe first- technique, this of simplicity the Despite . T stem cells and cells; however,human embryonic + T cells isolated from human peripheral peripheral human from isolated cells T b This protocol describes gene knockout knockout gene describes protocol This 6 4 . 62,64,70,7 2 . These conditions are are conditions These . c 4OO 8 ) 8 7 . RNP can be 62,6 4 .We - - - -

© 2015 Nature America, Inc. All rights reserved. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • REAGENTS M solution reaction the cells, activity. to application gene-editing direct their TALEN-mediated Before maximum yield to ( Cys-(Npys)- with react concen to and allowed are purified proteins TALEN Once trated, cytosol. the into entry their after poly-Arg and conditions, activity cell-penetrating with TALEN proteins endow to charge positive effector TAL poly-Arg individual thiol-reactive of with repeats conjugation that showed cells viously mammalian into proteins TALEN of delivery TALEN protein delivery. culture cell in in activity their enhance to order conditions culture shock’ ‘cold transient to exposed be also must proteins ZFN internalized, Once agent. protein-stabilizing and scaffold, zinc-finger the stabilize to supplements: ZnCl • d HiScribe T7 transcription kit(New Biolabs, England T7transcription HiScribe E2040S) HEPES sodiumsalt(Sigma-Aldrich, cat. no. H3784-100G) (Life Technologies, calcium andmagnesium HBSS with cat. no. 14025-092) Glycerol (Sigma-Aldrich, cat. no. G5516-100ML) GlutaMAX (Life Technologies, cat. no. 35050-061) Glucose (Sigma-Aldrich, cat. no. G7021-100G) Ficoll-Paque Plus (GE Healthcare Life Sciences, cat. no. 17-1440-02) FBS (Life Technologies, cat. no. 16000-044) PCRsystem (Roche,Expand high-fidelity cat. no. 11732641001) storedand itshouldbe under appropriate andhandled conditions. Ethanol (Sigma-Aldrich, cat. no. E7023) cat. no. 05892791001) EDTA-free complete protease inhibitor cocktail (Roche, tablet cat. no. 19052) humanEasySep CD4 cat. no. 14190-144) Dulbecco’s PBS, nocalcium, (DPBS; nomagnesium Life Technologies, DMEM (Life Technologies, cat. no. 10566-016) dNTPs (New Biolabs, England cat. no. N0447S) DMSO (Sigma-Aldrich, cat. no. D2650) DMEM/F12 medium (Life Technologies, cat. no. 11330) d Cys (Npys)-( CutSmart buffer (New Biolabs, England cat. no. B7204S) tested for each batch. batches andsuppliers. cell conditions dissociation Optimal shouldbe  IV(Life Technologies,Collagenase type cat. no. 17104-019) Chloroform (Sigma-Aldrich, cat. no. C7559-5VL) CD3/CD28 human T-cell activation beads (Life Technologies, cat. no. (BDBiosciences,Bacto yeast extract 11131D) cat. no. 212750) (BDBiosciences,Bacto tryptone cat. no. 211705) (BDBiosciences,Bacto agar cat. no. 214050) Kanamycin (Sigma-Aldrich, cat. no. 70560-51-9) Agarose (Bio-Rad, cat. no. 161-3100) Accutase (Stem Technologies, Cell cat. no. 07920) AccuRuler RGB protein marker (BioPioneer, cat. no. PM-001) DNA dye, loading 6×(New Biolabs, England cat. no. B7021S) buffer,TBE running 5×(Life Technologies, cat. no. LC6675) Laemmli protein dye, loading 2×(Bio-Rad, cat. no. 161-0737) TAE buffer, running 10×(Life Technologies, cat. no. AM9869) DNA ladder, (New 100bp Biolabs, England cat. no. N3231S) ATER To ensure activity, ZFN proteins must be maintained with two with maintained be Tomust proteins ZFN activity, ensure , -Arg) l

CR -DTT (Sigma-Aldrich,-DTT cat. no. D0632) I T I I 7 CAL ALS 9 0 under conditions that were previously determined determined previously were that conditions under . These disulfide linkages are reversible under reducing reducing under reversible are linkages .disulfide These The quality of collagenase type IV can vary between IVcanvary collagenase type of quality The d -Arg) 9 peptide (AnaSpec, peptide cat. no. AS-61206) + 2 T-cell enrichment cocktail (Stem Technologies, Cell , which coordinates with Cys and His residues This protocol describes the CPP-mediated 9 is released from the TALEN proteins proteins TALEN the from released is !

CAUT 9 moieties imparts enough enough imparts moieties I ON l -Arg, which acts as a as acts which -Arg, Ethanol isflammable, 7 7 .

0 . We pre We .

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Cas9 RNP delivery. RNP Cas9 delivery. TALEN protein mum maxi achieve to necessary be may ratio peptide-to-protein the in differences of Because of optimization cell types, certain CPP-mediated uptake between inhibitor. protease of in presence (SFM) the medium serum-free with hydrox thoroughly mixed sodium and M 1 ide using pH physiological to neutralized is • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • for regenerative medicine applications. medicine regenerative for of cells modify to used levels be could it thus and hESCs, in highest modification the facilitates approach this delivery, protein HDR via substitutions ssODN recombinogenic highly with cells into delivered be RNPs can In addition, cations. deletions chromosomal targeted RNPs multiple in delivering for tandem inducing simultaneously described originally Kim by was that procedure optimized an resents rep it and RNPs, with (hESCs) cells stem embryonic human of T4 DNA (New ligase Biolabs, England cat. no. M0202S) SYBR Safe DNA gelstain(Life Technologies, cat. no. S33102) SURVEYOR mutation detection kit(Transgenomic, cat. no. 706021) phosphateSodium monobasic (Sigma-Aldrich, cat. no. S8282-500G) phosphateSodium dibasic(Sigma-Aldrich, cat. no. S7907-100G) hydroxideSodium (Sigma-Aldrich, cat. no. S8045-500G) chlorideSodium (Sigma-Aldrich, cat. no. S3014-500G) acetateSodium (Sigma-Aldrich, W302406-1KG-K) SepMate-50 column (STEMCELL Technologies, cat. no. 15460) RPMI 1640medium (Life Technologies, cat. no. 11875-119) Recombinant interleukin-2 (rIL-2; R&DSystems, cat. no. 202-IL-010) Technologies, cat. no. 13256-029) Recombinant human basicfibroblastgrowth factor (bFGF; Life DNAQuickExtract solution (Epicentre, extraction cat. no. QE0905T) QIAprep spinminiprep kit(Qiagen, cat. no. 27104) QIAquick kit(Qiagen, PCRpurification cat. no. 28104) QIAquick kit(Qiagen, gelextraction cat. no. 28704) Polypropylene gravity-flow purification column (Bio-Rad, cat. no. 732-1010) Poly- Plasmid: pET-28b (EMDBiosciences, cat. no. 69865-3) according to themanufacturer’s instructions. cat. no. 15593-031) Phenol:chloroform:isoamyl alcohol (25:24:1(vol/vol); Life Technologies, Penicillin-streptomycin (Life Technologies, cat. no. 15140-148) Nuclease-free water (Promega, cat. no. P1193) Nickel-nitrilotriacetic (Ni-NTA) acid agarose (Qiagen, cat. no. 30210) mTeSR1 5×supplement (STEMCELLTechnologies, cat. no. 05852) mTeSR1 (STEMCELLTechnologies, cat. no. 05850) Mouse anti-human CD4antibody, FITC-labeled (Abcam, cat. no. ab59474) cat. no. 11140-050) MEM non-essential solution, aminoacid 100×(Life Technologies, Matrigel, factor growth reduced (BDBiosciences, cat. no. 354277) Magnesium chloride hexahydrate (Sigma-Aldrich, cat. no. M2393-100G) l Knockout replacement serum (SR; Life Technologies; cat. no. 10828-028) cat. no. I5502-1G) Isopropyl- Imidazole (Sigma-Aldrich, cat. no. I5513-100G) recommended. human blood. vaccinated previously Being for hepatitis A andBishighly for humanobtained all samples. Always usegloves manipulating when Normal Donor Blood Program) Human Research Scripps mixed blood anticoagulant (The Institute’s with -Arginine ( -Arginine l -lysine solution (Sigma-Aldrich, cat. no. P4707-50ML) et al. et β - l d -Arg; Sigma-Aldrich, cat. no. A5006-100G) -1-thiogalactopyranoside (IPTG;-1-thiogalactopyranoside Sigma-Aldrich, 7 2 . This approach is highly flexible and capable of of capable and flexible highly is approach This . !

CAUT This protocol describes the nucleofection nucleofection the describes protocol This natureprotocols 7 I 8 ON 72–7 . Compared with both ZFN and TALEN and ZFN both with . Compared It istoxic swallowed or inhaled. if Handle it 4 ! for the introduction of single-base single-base of introduction the for

CAUT 7 2 I and multiplexed gene modifi gene and multiplexed ON Informed consent shouldbe

| VOL.10 NO.11VOL.10 protocol | 2015

1845 - - - -

© 2015 Nature America, Inc. All rights reserved. • • • • • • • • • • • • • • • • EQUIPMENT • • • enzymes Restriction • • • • • • lines Cell • • • Vectors andprimers • • • • • • Uppercase basesindicate restriction sites. T 1846 • able able XhoI SpCas9 rev NcoI SpCas9 fwd XhoI TALEN rev NcoI TALEN fwd XhoI ZFN rev NcoI ZFN fwd P ! β Zinc chloride (Sigma-Aldrich, cat. no. 229997-10G) Zero Blunt TOPO PCRcloning kit(Life Technologies, cat. no. K2800-40) Trypsin-EDTA, 0.05%(wt/vol; Life Technologies, cat. no. 25300-062) itisswallowedX-100 ishazardous or inhaled. if Triton X-100(Sigma-Aldrich, cat. no. T8787-50ML) T4 DNA reaction ligase buffer (New Biolabs, England cat. no. B0202S) protocol 10 ml, cat. no. 89130-898; 25ml, cat. no. 89130-900) International;Sterile serological pipettes (VWR 5ml, cat. no. 89130-896; Sterile 50-mlconical (BDBiosciences, tubes cat. no. 352070) Sterile 15-mlconical (Corning, tubes cat. no. 14-959-49D) S-450 sonicator (Branson Sonifier, cat. no. 15-338-553) Refrigerated microcentrifuge (5415R; Eppendorf, cat. no. 022621408) PCR thermocycler (MJResearch PTC-2000) cell 4D-nucleofector kit(Lonza,P3 primary cat. no. V4XP-3032) NanoDrop Scientific) 2000cspectrophotometer (Thermo Leica microscope (Leica, DFC340FX) Hemocytometer (Hausser Scientific, cat. no. 3200) XRsystem Doc Gel (Bio-Rad, cat. no. 170-8195) (Stem Technologies, Cell magnet EasySep cat. no. 18000) Avanti (Beckman, J-Ecentrifuge cat. no. 369001) cat. no. UFC901024; 30-kDaMWCO, cat. no. UFC903024) Amicon filter centrifugal units(Millipore, Ultra-15 10-kDaMWCO, Polystyrene round-bottom tubes, 5ml(Corning, cat. no. 352058) Petri dish, 10cm(Corning, cat. no. 25373-041) TBE gel, 10%, (Life wt/vol Technologies, cat. no. EC6275BOX) XbaI (New Biolabs, England cat. no. R0145S) XhoI (New Biolabs, England cat. no. R0146S) NcoI (New Biolabs, England cat. no. R0193S) accordance institutional regulations. with H9 human stem embryonic cells (hESCs) Chemically competent TOP10 cells (Life Technologies, cat. no. C4040-06) Chemically competent BL21 (DE3) cells (Life Technologies, cat. no. C6010-03) Jurkat Type E6-1(American Culture Collection, cat. no. TIB-152) TypeK562 (American Culture Collection, cat. no. CCL-243) He and RNPs here used uponrequest obtained canbe by contacting J.L. andmammalianexpressionBacterial vectors encoding theZFNs, TALENs Life Technologies) M13 forward primer, 20 Primers (

-Mercaptoethanol, 55mM( rimer name CAUT l

a (American Typea (American Culture Collection, cat. no. CCL-2) | VOL.10 NO.11VOL.10 1 1 I ON | Table

Primer sequences for nuclease cloning. ( β -ME is hazardous if itiscontacted-ME ishazardous or ingested. if 1 ; Scientific, ThermoFisher Invitrogen custom DNA oligos) | 2015 aaaCTCGAGttaatgatgatgatgatgatgggagccgcccactttgcgtttctttttcggggagccgcc aaaCCATGGatgggcagcagccccaagaagaagaggaaggtgggcggctccatggataagaaatactca aaaCTCGAGttaaaagtttatctcgccgttatt aaaCCATGGatgatgggtcatcatcatcatcatcacggtggcagcgactacaaagaccatgacggt aaaCTCGAGttaaaagtttatctcgccgtt aaaCCATGGatgggtcatcatcatcatcatcacggtggcagcccgaaaaaaaaacgcaaa S equence (5 µ M (5 | β natureprotocols -ME; Life Technologies, cat. no. 21985-023) ′ -GTAAAACGACGGCCAGT-3 ′ –3 ′ ! )

CAUT I ON ! Handle H9cells in

CAUT I ON ′ ; Triton

• • autoclave thesolution. Store thebuffer at4°Cfor upto 6months. sodiumphosphate dH monobasic in1literof of phosphateSodium buffer (200mMstock solution, 1liter) it at−20°Cfor upto 6months. Prepare 1-mlaliquots; sterilize thesolution usinga0.22- (1Mstock solution,DTT 10ml) solution atroom temperature for upto 6months. Adjust thepHto 7.4andsterilize it usinga0.22- l temperature for upto 1 year dH 100 mlof Imidazole (2Mstock solution, 100ml) up to 1year. dH of ZnCl temperature for upto 1 year. dH 1 liter of MgCl Store itatroom temperature (25°C)for upto 1month. Ethanol (75%, vol/vol, 100ml) 4 °Cfor upto 1month calcium andmagnesium. HBSSwith Store IVin2mlof thesolution at type IV(1,000×stock solution) Collagenase type autoclave thesolution. Store itat4°Cfor upto 6months. salt indH HEPES (200mMstock sol Store itat4°Cfor upto 1month. Sterilize itby autoclaving, to LBagar asterile Petri andpipettedish. 22mlof plates (1liter)LB agar then autoclave the solution. Store it at 4 °C for up to 1 month. 10 g NaClof LB medium (1 liter) REAGENT SETUP • • • • • • -Arg (1Mstock solution, 100 ml) Tissue Scientific, culture incubator (Thermo cat. no. 50116050) Tissue culture (Labconco, hood cat. no. 332391120) 24 well, cat. no. CLS3527-100EA; 96well, cat. no. CLS3596) Tissue culture plate (Sigma-Aldrich; sixwell, cat. no. CLS3516-10EA; Sterile 1.5-mlmicrocentrifuge (Eppendorf, tubes cat. no. 022431021) 0.45 Low-protein-binding filter (Millipore; 0.22 Flat-cap PCRtubes, 0.2ml(Axygen, cat. no. PCR-02-C) Tris-glycine gel, 4–20%, (Life wt/vol Technologies, cat. no. EC6028BOX) X unit, cat. no. AAF-1002X) 4D-Nucleofector system (Lonza; core unit, cat. no. AAF-1002B; 2 2 2 µ (9mMstock solution, 1liter) (1Mstock solution, 100ml) O andautoclave thesolution. Store itatroom temperature for m, cat. no. SLHP033RS) 2 O. Adjust thevolume to 1liter andthepHto 8.0, andthen 2 2 O andautoclave thesolution. Store thesolution atroom in dH 2 O andautoclave thesolution. Store thesolution atroom 2

O. Adjust the volume to 1 liter and the pH to 7.5, and Dissolve 10 g 5 Bacto tryptone, of g yeast of and extract

Add 15 g of Bacto agar to 1 liter of LBmedium. to Bactoagar Add 1liter of 15gof ution, 1liter) .

Mix 25 ml of dH Mix 25mlof

Dissolve 1.54 g of DTT in 10 ml of dH in10mlof DTT Dissolve 1.54gof the SpCas9-encoding gene Reverse primer used to PCR-amplify the SpCas9-encoding gene Forward primer used to PCR-amplify TALEN-encoding genes Reverse primer used to PCR-amplify TALEN-encoding genes Forward primer used to PCR-amplify ZFN-encoding genes Reverse primer used to PCR-amplify ZFN-encoding genes Forward primer used to PCR-amplify P

Dissolve 17.4 g in 100 ml of dH Dissolve 17.4gin100ml of urpose

Dissolve 20.3 g of MgCl Dissolve 20.3gof Dissolve 1.23 g of ZnCl Dissolve 1.23gof

Dissolve 13.6 g of imidazoleDissolve in 13.6gof Dissolve 52.1 g of HEPESDissolve sodium 52.1gof µ

m, cat. no. SLGV013SL; Dissolve 1 g of collagenaseDissolve 1gof µ 2 O. Adjust thepHto 5.5and 2 m filter. Store the O with 75 ml of ethanol. 75mlof O with µ m filter, andstore

Dissolve 23.4g 2 2 ·6H in1liter

2 O in

2 O.

© 2015 Nature America, Inc. All rights reserved. cells onice for 30s, and transfer the cellsuspension toaculture tubecontaining 1ml of LBmedium. Incubate the cells 7| 6| 1 insert-to-vector molar ratio of 6:1ina10- 5| as described inStep2. 4| Visualize DNAbyagarose gel electrophoresis using SYBR Safe, asdescribed inStep2. of 10×CutSmart bufferand 20Ueachof the restriction enzymes NcoIand XhoI. Incubate the reaction at37°Cfor 3h. 3| ? pink or red after gel slice is dissolved, add 3 M sodium acetate dropwise until the solution becomes yellow.  Determine DNAconcentration byNanoDrop spectrophotometry measuring absorbance at260nm( gel through aQiagen spincolumnaccording tothe manufacturer’s instructions, and eluteDNAusing 30 500 1% (wt/vol) agarose gel. Run the gel at 100 volts (V) for 45 min. Cut the desired band from the agarose gel, and dissolve it in 2| 55 °Cfor 30sand 72°Cfor 2minfor ZFNs, 4minfor TALENs or6minfor Cas9,and final extension at72°C for 10min. water upto50 Expand high-fidelity bufferwithMgCl encode 5 1| E PROCE dH into 799mlof 1MMgCl 1 mlof Storage buffer (1liter) buffer. Store thebuffer at4°Cfor upto 6months. Elution buffer (1liter) buffer. Store thebuffer at4°Cfor upto 6months. Wash buffer B(1liter) buffer. Store thebuffer at4°Cfor upto 6months. Wash buffer A (1liter) solution at4°Cfor upto 24h. protease inhibitor cocktail to thebinding buffer before use. Store the Lysis buffer (1liter) up to 6months. the volume to 1liter andautoclave thesolution. Store itat4°Cfor 9 mMZnCl glycerol dH into 799mlof 1MMgCl 1 mlof Binding buffer (1liter) 0.22- protease inhibitor dH cocktail in2mlof tablet Protease inhibitor cocktail (25×stock solution) using a0.22- Cys1 mgof (Npys)-( Cys (Npys)-( autoclave thesolution. Store itatroom temperature for upto 1year. acetate anhydous powder dH in100mlof acetateSodium (3Mstock solution, 100ml) with shaking at 250r.p.m. for 1hat37°C. xpression vector construction

TROUBLES µ

CR l of 10×T4ligase buffer. Incubate the reaction atroom temperature for 1h. µ µ Keep the cellsonice for 30 min.Heat-shock the mixture at42°Cfor 60susing awaterbath,and then incubate the Thaw 100 Ligate the purified nuclease-encoding gene(s) from Step4withthe digested pET-28 vectorfrom Step4using an Purify the digested DNA by gel extraction, and then determine the DNA concentration using NanoDrop spectrophotometry, Digest pET-28 and nuclease-encoding gene(s) from Step2inseparate 50- Mix the PCR products from Step 1 with 10 PCR-amplify the gene(s) encoding the ‘left’and ‘right’ ZFNand TALEN monomers orSpCas9 using primers m filter. Store itat−20°C for up to 1 week. I l of gel solubilization buffer QG (provided in the QIAquick gel extraction kit) for 20 min at 50 °C. Run the solubilized T D I URE CAL ′ 2 -NcoI and 3 stock solution and an additional 87.7 g of NaCl. stock solution 87.7gof andanadditional Adjust µ d m filter, andstore itat−20°C for up to 6months. -Arg)

H STEP OOT 2 2 2 µ stock solution, NaCl glycerol 29.2gof and100mlof stock solution, NaCl 29.2gof and100mlof O. For ZFNstorage buffer, 1M 100mlof add µ 9 l of chemically competent TOP10 cells on ice, and mix them gently with 10

peptide solution (2.3mMstock solution) peptide d l. Use the following cycling program: initial denaturation at95°Cfor 5min,30cyclesof 95°Cfor 30s, Add 1mM To ensure that the gel slice is completely dissolved, invert the tube every 5 min. If the solution becomes -Arg) I

N Add 17.5 ml of 2MimidazoleAdd binding to 1liter 17.5mlof of Add 2.5 ml of 2MimidazoleAdd binding to 1liter 2.5mlof of Add 100 ml of 200mMHEPESAdd stock solution, 100mlof Dissolve 20.4 g of imidazoleDissolve binding into 1liter 20.4gof of Add 100 ml of 200mMHEPESAdd stock solution, 100mlof G 2 ′ O. For ZFNbinding buffer, 10mlof add -XhoI restriction sites( 9 peptide in250 peptide β -mercaptoethanol ( 2 ● O. Adjust thepHto 5.2and

T l of dH l of Dissolve 24.6 g of sodium Dissolve 24.6gof 2 IMI 2 O, andsterilize itusinga , 0.2

Dissolve one Roche N 2 G β µ O. Sterilize thesolution ~5d -ME) and1× µ l reaction containing ~50 ng of digested DNA,400Uof T4DNAligase and M of eachprimer, 0.2mMdNTPsand 0.5 T µ able l of 6× DNA loading dye, and resolve the PCR by gel electrophoresis using a

l Dissolve 1 -Arg.

). Carryouta50-

Poly-lysine coating EQUIPMENT SETUP at −20°Cfor upto 6months. mTeSR1400 mlof medium. Store themedium at4°Cfor upto 2weeks or Complete mTeSR1 medium the medium to 37°Cbefore use. DMEM/F12 medium. Store themedium at4°Cfor 1month. Prewarm GlutaMAX, 55mM 1.5mlof replacement, 100×non-essential solution, aminoacids 6mlof 6mlof Complete DMEM/F12medium stored at4°Cfor 3months. Prewarm themedium to 37°Cbefore use. penicillin-streptomycin DMEMmedium. to 500mlof solution This canbe Complete DMEMmedium storedbe at4°Cfor 3months. Prewarm themedium to 37°Cbefore use. penicillin-streptomycin RPMI1640medium. to 500mlof solution This can Complete RMPImedium Store thesolution at4°Cfor upto 2weeks. For Cas9 protein storage buffer, stock solution. 1MDTT 1mlof add into aT25flask. Store theflaskat4°C for up to1week. Incubate theflask tosolutionMatrigel 5%(vol/vol) 3mlof DMEM/F12medium andadd with Matrigel-coated T25flask for 2 h before use to ensure efficient cell attachment. be stored at 4 °C for up to 1 month. poly-lysine–treated plates at room temperature for 2 h. Coated plates can the solution by and aspiration wash it 0.5 twice with ml DPBS. of Dry 24-well culture plate, and incubate the plate at 37 °C for 1 h. Remove at room temperature for 2himmediately before seeding. µ l reaction using 5ng of templateDNA,5 µ l reactions containing 2

Add 250 ml poly-lysine solution of to each a well of natureprotocols

Add 50 ml of FBS and 5 ml of 100 U/ml of 100U/mlof FBSand5mlof Add 50mlof µ

One or 2dbefore H9hESCseeding, dilute

Add 50 ml of FBS and 5 ml of 100 U/ml of 100U/mlof FBSand5mlof Add 50mlof l of high-fidelity DNApolymerase with β Add 100 ml of mTeSR1Add 100mlof supplement (5×)into µ -ME and2.4 l of the ligation reaction from Step 5.

Add 120 ml of KnockOutAdd serum 120mlof 

CR I A T I 260 µ CAL g of bFGF into 466 ml of bFGFinto 466mlof g of

| ) ×50 VOL.10 NO.11VOL.10 Allow the plates to dry µ µ g of DNA,5 l of water. µ 62,70,7 protocol g/ml. µ | l of 10× 2 2015 that

1847

© 2015 Nature America, Inc. All rights reserved. 21| protein-bound slurryinto the columnwiththe bottom capattached. 20| incubate itonarotisserie at4°Cfor 30min. 19| Filter the supernatant through a0.45- 18| ? may be necessary for complete lysis.  off intervals. 17| protease inhibitor cocktail should be added to the lysis buffer immediately before use.   16| protein activity.  Do not dry the cellpellets. 15| ? as a result of reduced toxicity from nuclease proteins and improved folding.  TALEN-expressing cultures atroom temperature for 4h,and Cas9-expressing culture at 18°Cfor 12h. 14| We recommend monitoring the optical density at 600 nm (OD   50 13| plate, and culture itovernight at37°C. 12| 100 ng of sequence-verified nuclease expression vectorfrom Step10. Transform the cellsas described inSteps7and 8. 11| E ?  and confirmplasmid identity byDNAsequencing. 10| and culture itovernight at37°Cwithshaking. 9| overnight at37°C. 8| 1848 wash bufferB. protocol xpression and purification of nuclease proteins

TROUBLES TROUBLES TROUBLES

PAUSE CR CR CR CR CR CR CR µ Purify the nuclease expression vectors using the Qiagen plasmid miniprep kit according to the manufacturer’s instructions, Remove the bottomcapand discard the flow-through. Wash the columnwith20ml of washbufferAand 5ml of Rinse anemptypolypropylene gravity-flow purification columnwith5ml oflysis buffer, and then transfer the Add tothe filtered lysate1ml of nickel–nitrilotriacetic acid (Ni-NTA) agarose (50%slurry in30%ethanol) and Centrifuge the celllysate at25,000 Lysethe cellsbysonication using the following settings: 50%poweroutput,and 2minprocess time with5son/10 Resuspend the cellpelletfrom Step15with20mlof lysisbuffer. Transfer the suspension toafresh collection tube. Afterinduction, collectthe cellsbycentrifugation at5,000 Induce protein expression once the culture reaches anOD The following day, transfer 20mlof the overnight starterculture from Step12into 1literof LBmedium containing The following day, inoculate 20mlof LBmedium containing 50 Aftersequence verification, thaw50

g/ml kanamycin, 200mMNaCl and 0.2%(wt/vol)glucose. The following day, inoculate 4mlof LBmedium containing 50 Spread 100 | I I I I I I I VOL.10 NO.11VOL.10 T T T T T T T I I I I I I I CAL CAL CAL CAL CAL CAL CAL

H H H I STEP STEP STEP STEP STEP STEP STEP NT OOT OOT OOT Cell pellets can be stored for up to 1 week at −20 °C, or for up to 1 month at −80 °C with no loss in nuclease µ Avoid overheating the solution by keeping the cells on ice during sonication. Multiple rounds of sonication Insufficient resuspension of the cell pellet will negatively affect the efficiency of sonication. For ZFN protein expression only, ensure that the lysis buffer contains 90 Protein yield is improved markedly by culturing cells at reduced temperatures after induction, probably Induction conditions are highly variable, and they depend on the stability of the protein being expressed. For ZFN protein expression only, supplement the medium with 90 Verify the sequence identity of each plasmid by DNA sequencing. Mutations can reduce nuclease activity. | l of the bacterial cellculture from Step7onanLBagarplatewith50 I I I 2015 N N N G G G | natureprotocols µ m low-protein-binding filter. g for 30minat4°Cand transfer the supernatant into afresh collection tube. µ l of chemically competent BL21(DE3)cellsonice and mixthem gently with ●

T IMI N G 600 600 ~6 d ) ) every 30 min until an OD g of 0.8with0.1mMIPTG. Afterinduction, culture ZFN-and for 10minat4°C.Decant and discard the supernatant. µ µ g/ml kanamycin withone colony from the LBagarplate, g/ml kanamycin withone colony from the LBagar µ M M ZnCl µ g/ml kanamycin and incubate it 600 µ 2 M M ZnCl . of 0.8 is reached. 2 . β -ME -ME and

© 2015 Nature America, Inc. All rights reserved. ( internalization and (option C)nucleofection, respectively. 27| P  to help maintain protein stability, standard freezing at −80 °C is also acceptable.  using liquid nitrogen and store them at−80°C. 26| ? (see Step27C(i–xii)). it atroom temperature for 1h.For 25| ? to ~40 acid assay (BCA)  low-protein-binding filter. proteins and RNPs).Filterthe protein using a0.22- MWCO for ZFNproteins and a30-kDaMWCOfor TALEN an Amicon ultra-15 centrifugal filterunit(usea10-kDa buffer, and concentrate the proteins to400–800 24| the highest purity. (wt/vol) Tris-glycine gel. Combine the fractions with dye. Boilat95°Cfor 10minand resolve ona4–20% eluted protein with5 23| precipitation. protein stability. Failure to add l  fractions with0.5mlof elution buffer. 22| (viii) -Arg to each fraction immediately after elution to enhance A rotein delivery (vii)

(iii) TROUBLES TROUBLES (vi) Discard the supernatant and repeat the washstepuntil the supernatant becomes transparent. (iv) Centrifuge the sample at1,200

(ii) ) ZF PAUSE (v) CR CR CR (i) Introduce ZFNs, TALENs and Cas9RNPsinto cellsvia (option A)direct application, (option B)CPP-mediated Add 250 Determine nuclease protein activityby Buffer-exchange the elutedfractions withstorage Analyze the fractions bySDS-PAGE. Mix5 Elutethe nuclease protein tentimes into separate I I I the Ficoll-Paque layerinthe SepMate column. glycophorin A. tetrameric antibody complex thatrecognizes CD8, CD14,CD16,CD19,CD20,CD36,CD56,CD66b, CD123,TCR  the cellsatroom temperature for 10min. T T T to a5mlround-bottom conical tube. Add 60 Resuspend the mononuclear cellsin1.2mlof DPBSwith2%(vol/vol)FBSand 1mMEDTA, and transfer the cells By using apipette, transfer the mononuclear celllayertoanew 50-mlcanonical tube, and washitwith40mlof Add 15mlof Ficoll-Paque into aSepMate-50 column, and then carefully pipette34mlof dilutedbloodsampleonto Dilute the bloodwithanequal volume of completeRPMI 1640medium using aserological pipette. Mixit well. Obtain 100mlof blood, mixed withthe anticoagulant heparin, from aqualified supplier. ? complete RPMI1640medium. Centrifuge the cellsat 300 N µ I I I

CAL CAL CAL

protein delivery intoprimary TROUBLES M M and Cas9 protein to ~90 CR

PO I

H H I T STEP STEP STEP µ NT I OOT OOT µ CAL l of eachconcentrated protein from Step24into 1.5-mlmicrocentrifuge tubes. Flash-freeze the samples l of EasySephuman CD4 Nuclease proteins can be stored at −80 °C for at least 1 year without loss of activity. 8 0 Avoid repeating freeze-thaw cycles to prevent damaging proteins. Although flash-freezing is recommended The protein concentration should be assessed using a BSA standard curve by SDS-PAGE For ZFN proteins only, add 50 I I or Bradford

N N H STEP G G OOT µ l of 2×Laemmliprotein loading The human CD4 I N G 8 1 l assay. To ensure maximum dosage into cells, ZFN and TALEN proteins should be concentrated -Arg can lead to ZFN protein µ in vitro M M (15 g for 10minatroom temperature withno brake. + + Tcellenrichment cocktailto1.2mlof mononuclear cells. Mixwelland incubate C Tcellenrichment cocktailisolatesCD4 D4 cleavage assaysusing RNP, sgRNAmust be µ in vitro + g/

µ T µ cells l of

µ l). µ l l of 1 M l using cleavage. Prepare the reaction asdescribed in µ

●

T IMI

N G g ~5d T TALEN and RNPdo not require ZnCl for 10minatroom temperature. able able ZnCl BSA, 1 mg/ml Target site–encoding PCR amplicon CutSmart buffer C Nuclease-free water ZFN, TALEN or RNP l -Arg, 1 M omponent 2 , , 900 2 2 |

In In vitro µ M + cleavage assay reaction. Tcellsbynegative selection using a natureprotocols 2 and in vitro l -Arg. –transcribed and purified

T able per sample Up Up to 20

| A VOL.10 NO.11VOL.10 200 200 ng mount 2 2 2 2 2 2 2 2 7 µ µ µ µ µ 9 and incubate l l l l l , , or bicinchoninic µ

l protocol

100 100 to 1 nM Titrate from γ 0.1 0.1 mg/ml | 2015 10 10 ng/ / 100 100 mM 90 90 δ Final and 1× µ

| M

µ 1849

1850 (xvii) (xiii) protocol B (xix) (xvi) (xiv) (xii) (vii) (xx) (xv) Incubate the cellsat37°Cfor 1h. (iii) (vi) (iv) (xi) (ix) ) (v) (ii) (x) (i) CPP

| VOL.10 NO.11VOL.10 Proper cell-to-beadratio iscrucial for cellactivation.  CO CD4 of CD3/CD28human Tcellactivation beadsand 50Uof rIL-2for stimulation and expansion. Transfer the remaining Add 1×10 nuclease assay(Step28)isready tobeperformed.  Incubate the samplesat65°Cfor 15min,followed byincubation at98°Cfor 15min. Activated cellsexpand insize.  supernatant and washthe cellsonce withserum-free DMEMmedium. At 48hafteractivation, collectthe cellsbycentrifugation at300 of QuickExtract DNAextraction solution. ? cells with500 low levelsof cytoxicity couldbeobservedathigh protein concentrations. observed using ZFNprotein concentrations between0.5  with 90 Cetrifuge the cellsat300 Collect the cellsbycentrifugation at300 Resuspend the cellsin250 recommended for achieving optimal rates of mutagenesis.  Incubate the cellsat30°Cfor 24h. Replace the medium withcomplete DMEMand incubate the cellsat30°Cfor 24 h. Insert the 5-ml conical tube containing the magnetic bead mix into the EasySep magnet, and allow it to stand for 5 min. for 2minatroom temperature. observed afterthe conjugation reaction. Precipitates canberemoved bycentrifuging the reaction solution at5,000  serum-free DMEMmedium. Adjust the pHof the reaction byadding 7.5 internalization.  Gently remove SFMand apply the entire TALEN protein solution onto the cells. Incubate the cellsat37°Cfor 2h. ? instructions. Evaluate the purityof isolatedCD4 hemocytometer. Stainthe cellsusing FITC-conjugated anti-human CD4antibody according tothe manufacturer’s  room temperature for 5min. At 24hafter incubation, transfer the cellsto37°Cand incubate them for an additional 24h. Before protein treatment, remove the medium from eachwell,and wash the cellsonce byadding 500 At 24hafterincubation, transfer the cellsto37°C and incubate them for anadditional 24h. Gently invertthe tubeand transfer the unbound cellsinto anew 5-mlconical tube. Count the cellsusing a Mix the magnetic beadsthoroughly and then add 120 Wash the cells once withDPBSand isolate the cellsfor the QuickExtract procecdure, as described inStep27A(xix). Seed HeLa cellsonto a24-wellplateatdensity of 1×10 ? internalization and genome-editing activity.  Incubate the cellsfor 2h atroom temperature. peptide and 1×complete protease inhibitorcocktail in 100mMsodiumphosphate buffer, pH5.5,ina75- At 24hafterseeding, incubate 3.3

-mediated delivery of

TROUBLES

2 TROUBLES CR PAUSE CR CR CR CR CR CR CR . Cellscanbecultured under these conditions for upto1week. + Tcellstoa10-cmdishcontaining 10mlof completeRPMI1640medium, and maintain them at37°Cwith5% I I I I I I I I T T T T T T T T I I I I I I I I CAL CAL CAL µ CAL CAL CAL CAL CAL

M ZnCl PO 6 | CD4 2015

H I H STEP STEP H STEP STEP STEP STEP STEP STEP NT OOT OOT OOT µ l of completeRPMI1640medium containing 50U/mlrIL-2. + Samplescanbeheld at4°Cfor upto1weekorstored indefinitely at−80°Cuntil the Surveyor 2 | Tcellsinto one wellof a24-wellplatecontaining 1mlof completeRPMI1640medium with25 and 100mM Activation of CD4

Activate Tcellsfor 24–72h.Prolonged activation candecrease genome editing efficiency. Lackof Transient incubation at30°Cenhances the activityof internalized ZFNproteins, and itis Depending onthe purity of the TALEN proteins and the quality of the peptide, precipitation may be Incontrast toZFNproteins, TALEN proteins should beincubated withcellsfor ~2hfor efficient Magnetic beadscanprecipitate, and they should bevortexed before use. The molar ratio of Arg I natureprotocols I I N N N G G G TALEN g l for 10minatroom temperature, discard the supernatant and resuspend the cellsin100

-Arg willmarkedly decrease the genome editing efficiency. Maximum modification is µ

l of serum-free DMEMcontaining 2 l proteins -Arg. + µ

Tcellsisrequired for achieving maximum ZFN-mediated gene modification rates. M each‘left’and ‘right’ TALEN proteins with100 9 g ● -CPP toTALEN protein must bebetween8:1and 15:1for efficient cellular for 10minatroom temperature and discard the superntant. Resuspend the

µ IMI l of 1Msodiumhydroxide, and mixthe solution wellwith175

+ N Tcellsbyflowcytometry, as described byGajand Liu G ~4d µ l of the beadstothe cells. Mixwelland incubate the tubeat µ M and 4 5 cellsperwell. µ M eachof ‘left’and ‘right’ ZFNproteins supplemented g µ

for 10minatroom temperature, discard the M. Depending onthe purityof ZFNproteins,

M Cys-(Npys)-( µ d 6 -Arg) l of SFM. 7 .

l reaction. 9 µ

l of

µ µ l g l

( (xviii) (xvii) C (xiii) (viii) (xix) (xvi) (xiv) (xii) (vii) (xx) (xv) (iii) ) (xi) (ix) (vi) (iv) (x) (v) (ii) (i) E lectroporation of  ? sgRNA degredation.  spectrophotometry measuring the Dissolve the pelletin20 ethanol (75%(vol/vol))toremove residual salt. followed by200 Centrifuge at12,000 Add 80  mix. Incubate the reaction at37°Cfor 16h. containing 1×reaction buffer, 1 Transcribe the sgRNAby‘run-off’reaction using the HiScribeT7transcription kit.Carryouta20- Add 200 transcription reaction. Mixwelland incubate the reaction for 15minat37°C. Centrifuge the tubeat12,000 Add 2.5volumes of 100%ethanol tothe chloroform-extracted sgRNA.Store the mixture at−20°Cfor 12h. Centrifuge the tubeat12,000 After 16h,add 70 Centrifuge the tubeat12,000 ?  complete mTeSR1 medium tostopthe digestion.  stop the digestion. with 0.5mg/ml collagenase typeIVat37°Cfor 30min. Add 2mlof completeDMEM/F12medium tothe wellto Dissociate the cellsinto asingle-cell suspension bycarefully pipetting the cellsupand down 4–6times. Add 5mlof After one passage onMEFfeeder cells(5–7d),treat H9 hESCswith0.5mlof DMEM/F12 medium supplemented modification, as described previously by Pruett-Millerand Davis containing homology arms oneachside of the intended modification thatare atleast500bpinlength as described This is achieved by designing a forward primer to include a T7 promoter sequence immediately upstream of the sgRNA.  30 cyclesof 95°Cfor 30s, 55°Cfor 30sand 72°Cfor 40s, and final extension at72°C for 10min. DNA polymerase withwaterto50 5 (Optional) Perform this steponlyifsingle-base genome editing isdesired. Prepare adonor plasmid for HDR Repeat the chloroform extraction procedure once more, asdescribed inStep27C(v–vii). PCR-amplify sgRNAfrom amammalian expression vector( incubation with1mlof Accutase for 5minat37°C. conical tube, carefully remove the medium bypipetting. feeder layerin2mlof completeDMEM/F12medium. After 2–3dof incubation under feeder-free conditions, remove the medium and detach the H9cellsfrom Matrigel by Gently pipettethe cellsand transfer them toasterile15-mlconical tube. Once the cellssettleatthe bottomof the Acquire H9hESCsfrom aqualified supplier. Maintain the cellsinasix-wellplateon mouse embryofibroblast (MEF) in vitro  NanoDrop measuring the Purify the PCRproducts bygel extraction, asdescribed inStep2,and determine the DNAconcentration using a H9 cellsinthe following steps.  remove the supernatant medium. Wash the cellstwice withcompletemTeSR1 medium. Allowthe cellstosettleatroom temperature, and carefully Resuspend the H9cellswith completemTeSR1 medium and transfer them toasix-wellplateprecoated withMatrigel.

TROUBLES

PAUSE CR CR CR CR CR CR CR l of 10×Expand high-fidelity bufferwithMgCl I I I I I I I T T T T T T T I transcription steps. I I I I I µ I CAL CAL CAL CAL CAL CAL CAL µ l of nuclease-free waterand 20

PO l of chloroform toextract residual phenol.

I H STEP STEP STEP STEP STEP STEP STEP NT OOT OOT PurifiedsgRNAcanbestored at−80°C for atleast1yearwithout loss of activity. µ C Resuspending the pelletinnuclease-free waterand storing itat−80°Ciscrucial for preventing The PCR product must contain a T7 promoter sequence for downstream Short RNArequires a16-hreaction time for maximum transcription. Single-cell suspension iscrucial for achieving maximum nucleofection efficiency. Celldetachment canbe monitored under amicroscope. I Isolation of high-quality DNAtemplatefor RNAtranscription reaction isessential for subsequent Two washstepsare neccessary toremove the MEFs, which may interfere withthe nucleofection of l of phenol-chloroform-isoamyl alcohol solution, and vortex. I as9 N

µ N

G g l of nuclease-free water, 10 G for 30minat4°C.Remove the supernatant and discard it,and then add 500

RNP µ A 8 l of nuclease-free water. Determine the sgRNAconcentration using NanoDrop 260 intohumanstemcells 2 , orssODNscontaining atleast40nt of homology on eachside of the intended ×50 g g g for 5minat4°C,remove the supernatant and air-dry the pellet. for 5minat4°C,and transfer the supernatant toanew microcentrifuge tube. for 5minat4°C.Transfer the supernatant toanew microcentrifuge tube. µ

g of purifiedPCRproduct, 7.5mM of eachdNTPand 1.5 A µ 260 µ l. Use the following cycling program: initial denaturation at95°Cfor 5min, g/ml. 73,7 ×40 8 µ . l of 3Msodiumacetatetothe DNase I–treated transcription reaction

g/ml. Store the sgRNAat−80°C. µ

2 l of 10×DNase Ibufferand 2 , 0.2 ●

T µ IMI M of eachprimer, 0.2mMdNTPsand 0.5 B N ox G ~5d 1 ). Carryouta50- natureprotocols

µ µ l of DNase I(RNase-free) tothe l PCRusing 5ng of templateDNA, in vitro µ l of T7RNApolymerase

| VOL.10 NO.11VOL.10 transcription reactions. µ µ l reaction

l of high-fidelity µ l of ice-cold

protocol

| 2015

1851

T ( restriction fragment length polymorphism (RFLP)or(option C)sequence verification. 28| ofQuantification gene modification 1852 (xxiv) (xxiii) (xxii) able able dH DMSO (%) Template DNA Reverse primer ( Forward primer ( polymerase ( Expand high-fidelity buffer (10×) Expand high-fidelity dNTPs (10 mM) protocol A (xxi) (iii) (iv) (ii) ) (v) Quench the reaction with1.5 (i) 2 Determine the gene editing efficiency of the nuclease proteins via (option A)Surveyor nuclease assay, (option B) O S

| urveyor nucleaseassay VOL.10 NO.11VOL.10 the Surveyornuclease assay.  heteroduplexes.  ? the internal PCR. to 3 3 Incubate the reaction at42°Cfor 1h. Mix 10 Verify amplification byrunning 10 PCR-amplify the genomic target sitebynested PCR,asdescribed previously Denature and re-anneal 30 ? temperature and resuspend them in 50 at 37 °C. Quench the reaction with 5 ml of complete mTeSR1 medium. Cetrifuge the cells at 115 At 48 h after incubation, wash the cells once with DPBS and isolate the cells by incubation with 1 ml of Accutase for 5 min 4D-nucleofector systemusing the program CB-150. Transfer the cellsto16-wellNucleocuvette strips(included inthe nucleofector kit)and electroporate them using a the nucleofection volume (20  experimental planning. full range of RNPamounts indicated here support efficient gene editing. Ranges are giventoprovide flexibility for Step 27C(xxi))mixed with2–4 Optionally, for co-delivery withdonor plasmid orssODN,use2 cell solution, 3.6 at room temperature, and resuspend the cellpelletin22 at room temeprature for 10mintoform the RNP. Centrifuge 2×10 Mix 15 six-well platecontaining 2mlof mTeSR1 medium. Immediately afternucleofection, add 100

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Troubleshooting table. Random mutations Nonspecific amplification P single-cell single-cell suspension Difficulty generating a Low sgRNA yield TALEN conjugation reaction Precipitation during the from centrifugation Difficulty recovering cells Low CD4 Cell pellet is colored red Poor nuclease activity Low purity Slow filtration Insufficient cell lysis Slow bacteria growth High background roblem | 2015 + T-cell purity | natureprotocols

Incorrect protease treatment work. Degradation of sgRNA chloroform extraction did not reaction failed or phenol In vitro Low TALEN protein purity of the microcentrifuge tube Cells are attached to the side oversaturated with PBMCs The antibody cocktail mix was Red blood cell contamination misfolding Contamination or protein Protein contamination the filter Precipitated protein is blocking incorrect sonication settings High density of bacterial cells or Nuclease toxicity Insufficient vector digestion Polymerase-induced errors product amplification repeats are contributing to side Zinc-finger or TAL effector P ossible reason transcription

prevent nonspecific nuclease binding and cleavage. that NaCl is present in the culture medium to Culture cells at room temperature, and ensure conditions described in Step 1 positive clones before DNA sequencing using the cycling If problems persist, use colony PCR (CIP) to prevent recircularization Treat the vector with calf intestinal alkaline phosphatase recommended for maximum yield optimization for expression in problems persist. If synthesized, codon usage DNA polymerase. Synthesize the nuclease gene if Use a high-fidelity DNA polymerase, such as Phusion secondary expression vector Alternatively, subclone the nuclease gene from a concentration of each primer and >100 ng of template. amplification, or repeat the PCR using <0.2 Include ~2.5 M betaine to help prevent nonspecific S a a P1000 micropipette Treat H9 cells with fresh Accutase. Mix cells well using sgRNA from a qualified manufacturer degradation. If trouble continues, purchase synthesized sgRNA. Use RNase inhibitors to prevent potential sgRNA transcription. Consider using an RNA minikit to purify Increase the amount of DNA template used for TALEN proteins Perform conjugation reaction using high-quality Use a swing-bucket centrifuge rotor antibody cocktail mix Increase the incubation time of PBMCs with the Reduce the amount of blood used during purification. the mononuclear cell layer Avoid contacting the red blood cells when transferring obtained from ToolGen, Life Technologies or PNA BIO) from a qualified manufacturer (e.g., Cas9 protein can be If trouble continues, purchase purified nuclease protein buffers, or perform additional buffer exchange steps. reduce the salt concentration in the lysis and elution Purify proteins at 4 °C to promote native conditions, or ion exchange chromatography) as described Performpreviously additional purification steps (e.g., size-exclusion during centrifugation to prevent clogging Gently mix or pipette the protein solution every 20 min using lysozyme Dilute the cell suspension. Alternatively, lyse cells an OD Induce protein expression when the culture reaches olution 600 of 0.2–0.5 Escherichia Escherichia coli 9 0 to screen for µ M is

(continued)

in in vitro

© 2015 Nature America, Inc. All rights reserved. previously ‘M’ indicates cells treated with serum-free medium only. Arrows indicate expected cleavage products. The modification efficiency is calculated as described in HeLa, K562, Jurkat and CD4 DNA is calculated by dividing the density of the cleavage product by the density of the total DNA within the same lane. ( substrate. Active ZFN proteins cleave DNA at concentrations as low as 10 nM. The percentage of cleaved ‘right’ ZFN protein is indicated by ‘1 NLS’, ‘2 NLS’ and ‘3 NLS.’ ( bacterial lysate ( Figure 2 three-NLS ZFNproteins alsoshowed ~25%modification afterasingle treatment ( teins demonstrated >30% modification of the negligable effectonZFNprotein activity( contained one tothree tandem repeats of the SV40NLS( We expressed and purified ZFNproteins (~1 mg perliterculture) thatwere designed totarget the human Modification of the ANT B Step 28C, sequence confirming the presence of mutations: ~5 d Step 28B, RFLP assay to measure HDR efficiencies: ~2 d Step 28A, surveyor nuclease assay to measure modification frequencies: ~2 d Step 27C, nucleofection of Cas9 RNP into human embryonic stem cells: ~5 d Step 27B, conjugation of CPPs onto the surface of TALEN proteins and their delivery into human cells: ~4 d Step 27A, delivery of ZFN proteins into primary cells: ~5 d Steps 11–26, expression and purification of nuclease proteins from bacterial cells: ~6 d Steps 1–10, construction of nuclease bacterial expression vectors: ~5 d ● T in in 10/45 samples (22.2%) treated with two-NLS ZFN proteins. (dashes) and insertions (lowercase) induced by ZFN proteins are shown in nine sequences aligned to the wild-type (WT) cleavage site. Mutations were found (kDa) a a able able 28A(viii) 28A(i) 27C(xxiv) S MW 100 ox ) ) SDS-PAGE of one-, two- and three-NLS ‘right’ AAVS1 ZFN proteins purified from the soluble fraction of

tep 11 17 25 35 48 63 75 T IMI I 1 C AAVS1 , , construction of nucleases: ~4–14 d I 8 8

PATE ZFN protein N | 8 | 3 2 1 Modification of the 9 G

. . ( Troubleshooting table (continued). ZFN proteins at different concentrations with 100 ng of substrate DNA. Arrow indicates cut d D 8 ) ) Sequence analysis of the 8 Nonspecific amplification plate after nucleofection No H9 cells attached to the P efficiency Low genome-modification . . Molecular-weight (MW) standards are indicated. The number of NLS repeats within each RESULTS roblem NLS repeats AAVS1 + AAVS1 T cells treated once with 4 b locusby direct delivery of ZF 0 100 89 1 NLS locus by direct delivery of zinc-finger nuclease (ZFN) proteins. 90 50 27 10 AAVS1 100 95 2 NLS locus from CD4 91 50 32 10 cell-penetrating activity Inactivate nuclease or poor Improper PCR conditions stem cell dissociation Toxicity from nucleofection or P Fig. 2 ossible reason b 100 ) ) 95 AAVS1 3 NLS In In vitro µ 93 50 b M M of one-, two- and three-NLS ZFN proteins, as determined by the Surveyor nuclease assay. ). HeLa, K562and Jurkat cellstreated with4 31 10 + T cells treated once with 2 locusafterone protein treatment ( cleavage assay of one-, two- and three-NLS Cleavage (% ZFN protein(nM N Fig. 2 proteins ) a ) ). c In vitro

M 3 1 HeLa 27 2 activity activity of purified nuclease protein by is no activity, construct new nucleases. Confirm the Test nuclease activity by transient transfection. If there PCR primers PCR. Alternatively, design and order new genomic Identify the optimal annealing temperature by gradient than 5 °C below the melting temperature of the primers. Ensure that the annealing temperature is no more dissociated stem cells kinase (ROCK) inhibitor to enhance the survival of of RNP used in the nucleofection. Use rho-associated Increase the number of cells or decrease the amount Use the correct nucleofection solution and program. S blotting. If trouble continues, construct new nucleases assay. Visualize internalized nuclease protein by western cleavage analysis revealed thatNLSlength hada µ olution M M each of left and right two-NLS ZFN proteins. Deletions 30 3 M

Fig. 2 9 1 K562

natureprotocols 29 2

36 c 3 Fig. 2

). Consistent withaprevious study, d M c ). CD4 Jurkat µ 9 1 c M of three-NLS ZFNpro ) ) Frequency of 28 2 31 3

+ | Tcellstreated with VOL.10 NO.11VOL.10 CD4 M AAVS1 5 1 + Tcells 22 in in vitro 2 AAVS1 protocol 25 locus 3 modification | Modification (% NLS repeats 2015 cleavage 8 3

and

- | +80 +4 (×2) +4 –24 –11 –10 –8 –7 –4 WT

1855

) © 2015 Nature America, Inc. All rights reserved. nuclease assay.nuclease ( EMX1 previously as described calculated was products. efficiency The modification 15 of 15 composed complexes RNP with nucleofected cells K562 AAVS1 lane. same ( the within DNA of the total density over product of the density cleavage dividing by calculated is DNA of cleaved percentage DNA at concentrations as low as 5 nM. The RNP Active cleaves DNA. ng 100 of substrate with incubated concentrations different at RNP ( for sgRNA. size of the bands anticipated specific show transcripts RNA All sgRNA. of purified ( are indicated. standards (MW) Molecular-weight protein. SpCas9 of purified RNP. of Cas9 delivery ( direct by loci 4 Figure cells modified bysingle RNPs( of RNPsalsoshowed efficient multiplexed gene modifications, with noappreciacable decrease inactivitycompared with the nucleofected witheither 7.5or15 analysis confirmed thatRNPdisplayed robust cleavage activity( lysates and incubated withpurifiedsgRNAtargeting the human The SpCas9nuclease wasexpressed athigh yield (~1mg/l culture) and >90%purityfrom the solublefraction of bacterial Multiplex genemodification by direct delivery of after transient transfection of TALEN-encoded plasmid DNA( treated with2 with 2 containing anintegrated EGFPgene whose expression isrestored via TALEN-mediated cleavage. Treatment of reporter cells cells and tostimulate mutagenesis using apreviously described conjugated tothe CPPCys-(Npys)-( TALEN proteins designed torecognize the human Modification of thehuman from CD4 multiple NLSrepeats improved the cellulargene-editing efficiency of ZFNproteins. Sequence analysis of was calculated as described previously by the Surveyor nuclease assay. ‘M’ indicates cells treated with serum-free medium. Arrows indicate expected cleavage products. The endogenousmodification efficiency with 2 same lane. Arrow indicates expected cleavage products. ( over the density of total DNA within the by dividing the density of the cleavage product The percentage of cleaved DNA is calculated cleave DNA at concentrations as low as 10 nM. left and right target sites. Active TALEN proteins 100 ng of substrate DNA containing symmetrical at different concentrations incubated with cleavage assay of left and right TALEN proteins weight (MW) standards are indicated. ( designed to target human ( gene by cell-penetrating TALEN proteins. Figure 3 1856 previously as described calculated was products. efficiency The cleavage modification and 15 a c protocol ) ) ) SDS-PAGE of left and right TALEN monomers µ In vitro In g of sgRNA, as determined by the Surveyor nuclease assay. ‘M’ indicates cells treated with serum-free medium only. Arrows indicate expected cleavage only.cleavage medium expected indicate Arrows serum-free with treated assay. nuclease cells indicates by ‘M’ the Surveyor g as determined of sgRNA, EMX1 and

-1 -1 and | µ VOL.10 NO.11VOL.10 µ M TALEN proteins (P) or transfected with 200 ng of each TALEN expression vector (D). Error bars indicate s.d. (

µ g of sgRNA), as determined by the Surveyor nuclease assay. ‘M’ indicates cells treated with serum-free medium only. medium expected indicate Arrows serum-free with treated assay. nuclease cells indicates by ‘M’ the Surveyor as determined g of sgRNA), | | Modification of the human AAVS1 Modification of multiple genomic of multiple Modification M TALEN protein ledto~2%EGFP-positivecells( cleavage assay of assay cleavage + gene and twodifferent d CCR5 Tcellstreated with4 AAVS1 , e ) Frequency of ) Frequency -2 loci in K562 cells nucleofected with 15 with nucleofected cells in K562 -2 loci µ g gene in HeLa cells treated with 2 g of Cas9 protein and g or 7.5 of protein Cas9 ) Modification of ) the Modification µ -2 -2 ( M TALEN proteins revealed ~15%modification of the CCR5 gene, compared with~5% modification observed | 2015 e ) modification in ) modification CCR5 | CCR5

. Molecular- EMX1 natureprotocols b ) Agarose gel ) Agarose CCR5 -targeting -targeting ( a CCR5 d 8 ) ) SDS-PAGE b Fig. 4e 9 ) ) and . ) AAVS1 geneby cell-penetrating

µ In vitro

AAVS1

M of two-NLSZFNprotein confirmed the frequency of ZFN-induced mutations (

d -Arg) g of RNPrevealed >20%modification of the CCR5 gene, and >50% modification of

-1 locus in H9 human embryonic stem cells nucleofected with 30 with nucleofected cells stem in H9 human embryonic -1 locus

f sites( ). H9hESCsnucleofected withsingle RNPdemonstrated >30%modification of the µ 9 M TALEN proteins (P) or transfected with 200 ng of each TALEN expression vector (D), as determined ( b a (kDa) Fig. 3a b (kDa) a MW 130 170 100 135 180 245 MW c 53 70 93 11 17 20 25 35 48 63 75 ) The percentage of EGFP-positive cells, as determined by flow cytometry in reporter cells treated AAVS1 µ g of Cas9 protein precomplexed with 7.5 7.5 with precomplexed g of protein Cas9 CCR5 monomer C SpCas9 TALEN L as9 , b -1 and ). We testedthe abilityof the resulting TALEN proteins toenter the R gene were purifiedfrom bacteria (~0.5–1.0 mg/l culture) and RNP c Fig. 3 1 0 CCR5 TALEN AAVS1 Fig. 3 f EMX1 50 50 8 57 Left 8 5 0 . . ( c 8 22 AA CCR5 Fig. 4 proteins CCR5 4 11 10 ). Inaddition, analysis of DNAisolatedfrom HeLa cells M

f VS1-1 human embryonic kidney (HEK)293Treporter cell line <5 ) Frequency of multiplexed modification of ofthe modification ) Frequency multiplexed -2; R 1 d RN AA 20 61 50

VS1-2 ). Righ Clea P , NP (nM) 8 Fig. 4d 9 c EMX1 10 10 . v t ). Analysis of DNAisolatedfrom K562cells age (% AA M Cleavage (%) TALEN (nM VS1 d RN >50 and ) M -1 , P e EMX1 >50 7.5 ). K562cellsnucleofected withcombinations AAVS1 >50 15 ) µ g of each sgRNA, as determined by the Surveyor by the Surveyor as determined g of sgRNA, each c Modification (% RNP M

EMX1 EGFP-positive cells (%) 0 1 2 3 4 5 6 RN >50 loci( ( µ Delivery method g) R P M µ g of RNP complex (15 g (15 of complex RNP AA M Fig. 4a ) P e n VS = 3). ( >50 NP 1 -2 D M AA Modification (% >50 7.5 VS1 , d b d ) Modification of the >50 15 -1 ). Delivery method CCR5 In vitro AAVS1 P M g ) M and AA D 1 5 µ M >50 VS1 7.5 g of Cas9 protein g of protein Cas9 lociamplified cleavage AAVS1 RNP >50 -2 5 Fig. 2 15 33 Modification (% Modification (% Modification (% RNP

-1, or -1,

( d µ

g) ).

) ) ) © 2015 Nature America, Inc. All rights reserved. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.htm at online available is information permissions and Reprints are in available the details interests: CO performed the experiments; and T.G., J.L. and J.-S.K. wrote the manuscript. C.F.B. and J.-S.K. designed the research; J.L., Y.Y., N.W., S.S., S.K. and C.N.K. AUT Molecular graphics were generated by PyMol. Basic Science (IBS-R021-D1 to J.-S.K.) and ShanghaiTech University (to J.L.). C.F.B.), The Skaggs Institute for Chemical Biology (to C.F.B.), the Institute for This work was supported by the National Institutes of Health (DP1CA174426 to A in K562cells( modification atfrequencies of 44%or7.5%, respectively, 200 pmol of ssODNsgaverisetoHDR-mediated genome AAVS1 sgRNA toCas9protein. Finally, co-delivery of 15 are achieved using two-tosix-fold molar excess of AAVS1-1 locus( calculated as described in Step 28B(vi). Arrow indicates the expected cleavage product. HDR efficiency was amplicons were digested with XbaI to measure the frequency of HDR. are complementary to the outside of the homology arm sequences. PCR restriction site (white bar). Target locus was amplified with primers that sgRNA and 2 nucleofected with 15 with donor plasmid or ssODN. HDR of the Figure 5 cknowle

H PET Acc. Chem. Res. Chem. Acc. technologies. modification genome and regulation gene targeted of advent proteins. finger Biotechnol. Nat. 1156–1160(1996). domain. cleavage FokI to fusions finger nucleases. zinc-finger designed nucleases. zinc-finger designed with targeting cells. human in (2008). 5809–5814 using engineered zinc-finger nucleases. zinc-finger engineered using Genetics in mutagenesis and cleavage USA Sci. cells. mammalian in recombination homologous stimulates nucleases. Biochem. engineering. genome for methods Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting targeting gene stimulate nucleases Chimeric D. Baltimore, & M.H. Porteus, Y. Santiago, chromosomal Targeted D. Carroll, & K.G. Golic, M., Golic, M., Bibikova, endonuclease site-specific a of Expression M. Jasin, F.& P.,Smih, Rouet, programmable with engineering genome to guide A J.S. Kim, & H. Kim, nucleases. targetable with engineering Genome D. Carroll, CRISPR/Cas-based TALEN,and ZFN, C.F.III. Barbas, & C.A. Gersbach, T., Gaj, Gersbach, C.A., Gaj, T. & Barbas, C.F. III. Synthetic zinc finger proteins: the proteins: finger zinc Synthetic C.F.III. Barbas, & T. Gaj, C.A., Gersbach, Cys by recognition DNA C.O. Pabo, & L. Nekludova, S.A., Wolfe, J.C. Miller, zinc enzymes: restriction Hybrid S. Chandrasegaran, & J. Cha, Y.G., Kim, F.D.Urnov, gene Enhancing D. Carroll, & J.K. Trautman, K., Beumer, M., Bibikova, OR I -1-specific RNPand 2

CONTR | G G FI Modification of the AAVS1 locus by direct delivery of RNP dgm

µ 91 161

NANC 83 g of donor plasmid or 200 pmol of ssODN containing an XbaI I ents Nat. Rev.Genet. Nat. , 6064–6068 (1994). 6064–6068 , et al. et BUT et al. et , 409–439 (2014). 409–439 , , 1169–1175, (2002). et al. et Fig. I

I Fig. 4

47 Science AL Annu. Rev. Biophys. Biomol. Struct. Biomol. Rev.Biophys. Annu. A TALE nuclease architecture for efficient genome editing. genome efficient for architecture nuclease TALE A We thank S.J. Sirk for critical reading of the manuscript. ONS 29 Highly efficient endogenous human gene correction using correction gene human endogenous efficient Highly µ Targeted gene knockout in mammalian cells by by cells mammalian in knockout gene Targeted , 2309–2318 (2014).2309–2318 , l I g of Cas9 protein precomplexed with 15 , 143–148(2011)., . 5 NTERESTS

J.L., J.L., T.G. and C.F.B. conceived the study; J.L., T.G., ).

g 300 ). Maximum modification efficiencies

15 , 763 (2003). 763 , Drosophila , 321–334 (2014).321–334 ,

online online version of pape the The authors declare competing financial declare financial competing The authors Nature µ g of HDR-donor plasmid or Trends Biotechnol.Trends

AAVS1 435 using zinc-finger nucleases. zinc-finger using Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 646–651 (2005). 646–651 , -1 locus in K562 cells Science

31 , 183–212 (2000). 183–212 ,

300 , 397–405 (2013). 397–405 , r http://www.nature. . , 764 (2003). 764 , Annu. Rev.Annu. Proc. Natl. Acad. Natl. Proc. µ µ g of 2 g of

His

105 2

zinc ,

32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 14. 22. 21. 20. 19. 18. 17. 16. 15.

10 lineages. different from cells mammalian transformed cells. transfected of activity metabolic the in changes non-specific induces Nucleofection (2003). complexes. peptide-liposome-DNA cells. mammalian into DNA of delivery efficient promotes 92 and culture in cells into procedure. transfection DNA. plasmid with cells transforming stably for system transfection efficient highly cells. primary for method transfection (1987). DNA. with cells mammalian of transfection (2015).120–127 RGEN. and TALENZFN, nucleases: engineered for specificity, and fidelity. and specificity, (2013). 230–232 endonuclease. RNA-guided Cas9 the with cells human e00471(2013). Science 339 immunity. bacterial adaptive (2011). targeting. DNA for constructs effector-based TAL effectors. TAL by effectors. Maurisse, R. Maurisse, L.A. Pardo, & Stuhmer,W. J.R., Agarwal, A., F., Sanchez, Queiroz, de Mello V.P.Torchilin, A. Eguchi, O. Boussif, P.L.Felgner, a transfer: gene phosphate-mediated Calcium H. Okayama, & C.A. Chen, Efficient A.K. Bosserhoff, & R. Blindt, I., Breibach, N., Krott, A., Hamm, efficient the for P. Electroporation Berg, & H. Hayakawa, G., Chu, systems delivery future and Current Y.H. Kim, & J.Y. Chung, Q.U., Ain, Boch, J. Boch, Maggio, I. & Goncalves, M.A. Genome editing at the crossroads of delivery, delivery, of crossroads the at editing Genome M.A. Goncalves, & I. Maggio, in engineering genome Targeted J.S. Kim, & J.M. Kim, S., Kim, S.W., Cho, M. Jinek, L. Cong, P.Mali, M. Jinek, T. Cermak, recognition DNA governs cipher simple A A.J. Bogdanove, & M.J. Moscou, 276 , 9 (2010). 9 , , 7297–7301(1995)., AA ssODN donor Plasmid donor ssODN donor Plasmid donor AA , 823–826 (2013). 823–826 , , 26204–26210 (2001).26204–26210 , VS1 VS1

339 et al. et et al. et et al. et et al. et al. et RN locus Science et al. et et al. et et al. et , 819–823 (2013).819–823 , et al. et et al. et RNA-guided human genome engineering via Cas9. via engineering genome human RNA-guided P Biotechniques Multiplex genome engineering using CRISPR/Cas systems. systems. CRISPR/Cas using engineering genome Multiplex Breaking the code of DNA binding specificity of TAL-type III of specificity binding DNA of code the Breaking et al. et RNA-programmed genome editing in human cells. human in editing genome RNA-programmed in endonuclease DNA dual-RNA-guided programmable A Protein transduction domain of HIV-1 Tat protein Tat HIV-1 of domain transduction Protein Efficient design and assembly of custom TALEN and other other and TALEN custom of assembly and design Efficient A versatile vector for gene and oligonucleotide transfer transfer oligonucleotide and gene for vector versatile A Science

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in vitro in 40 nt 337 Tissue Eng. Tissue 44 + + Xbal Xbal , 816–821(2012). ,

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