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1National Creative Research Initiatives Center for Genome Engineering, Seoul National University, Seoul, South Korea. 2Department of Chemistry, Seoul National University, Seoul, South Korea. 3These authors contributed equally to this work. Correspondence should be addressed to J.-S.K. (jskim01@snu.ac.kr).

Received 20 November 2012; accepted 14 January 2013; published online 29 January 2013; doi:10.1038/nbt.2507

The clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system provides prokary-otes with adaptive immunity to viruses and plasmids1. When Cas9, a protein component in the type II CRISPR-Cas system, is complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), it forms a sequence-specific endonuclease that cleaves foreign genetic sequences to protect host cells. The crRNA is transcribed from a host genomic CRISPR element previously cap-tured from such foreign invaders. A single-chain chimeric RNA pro-duced by fusing crRNA and tracrRNA sequences can replace the two RNAs in the Cas9-RNA complex to form a single-guide-RNA:Cas9 endonuclease (sgRNA:Cas9)2. Thus, in contrast to the widely used genome-editing technologies based on zinc finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs), the spe-cificity of RNA-guided endonucleases (RGENs) can be customized by replacing a short synthetic RNA molecule without changing the protein component. Here we show that sgRNA:Cas9 can induce site-specific genome modifications in human cells at high frequencies.

We first tested the DNA cleavage activity of the Cas9 derived from Streptococcus pyogenes (Supplementary Fig. 1) in the presence or absence of a chimeric RNA in vitro (Supplementary Methods and Supplementary Table 1). To this end, we used recombinant Cas9 protein to cleave plas-mid DNA that contained a 23-bp target sequence from human CCR5, which encodes an essential co-receptor of HIV and is a potential target for the treatment for AIDS3. A Cas9 target sequence consists of a 20-bp DNA sequence complementary to the crRNA or the chimeric RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recog-nized by Cas9 itself (Fig. 1a)2. Cas9 cleaved the plasmid efficiently at the expected position only in the presence of the synthetic RNA and did not cleave a control plasmid that lacked the target sequence (Fig. 1b).

Next, we used an RFP-GFP reporter4 to investigate whether the sgRNA:Cas9 can cleave the target sequence incorporated between the

targeted genome engineering in human cells with the cas9 rna-guided endonucleaseSeung Woo Cho1–3, Sojung Kim1–3, Jong Min Kim1,2 & Jin-Soo Kim1,2

We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.

RFP and GFP sequences in human embryonic kidney (HEK) 293T cells (Fig. 1c). In this reporter, the GFP sequence is fused to the RFP sequence out-of-frame. GFP is expressed only when the target sequence is cleaved by site-specific nucleases, which causes small, frameshifting insertions or deletions around the target sequence by means of error-prone nonhomologous end joining repair of the double-strand breaks. We found that GFP-expressing cells were obtained at frequencies rang-ing from 5% to 7% in three independent experiments only when the cells were co-transfected with the in vitro transcribed chimeric RNA and a plasmid encoding Cas9, demonstrating that sgRNA:Cas9 can recognize and cleave the target DNA sequence in eukaryotic cells.

To test whether the RGENs could be used for targeted disruption of endogenous genes in human cells, we analyzed genomic DNA isolated from transfected cells using T7 endonuclease I (T7EI), an endonuclease that specifically cleaves heteroduplexes formed by the hybridization of wild-type and mutant DNA sequences5. We found that mutations were induced only when the cells were co-transfected with both Cas9 and RNA. Mutation frequencies estimated from the relative DNA band inten-sities were RNA-dosage dependent, ranging from 2.0% to 18% in three independent experiments (Fig. 2a and Supplementary Fig. 2a), on par with those obtained with ZFNs or TALENs at the CCR5 locus5–8. DNA sequencing analysis of the PCR amplicons corroborated the induction of sgRNA:Cas9-mediated mutations at the endogenous site (Fig. 2a).

Both ZFNs and TALENs are associated with off-target effects7,9, which may limit the utility and safety profile of these enzymes in gene or cell therapy. Furthermore, the repair of two concurrent double-strand breaks induced by these enzymes at on-target and off-target sites can give rise to genome rearrangements10,11. The most important off-target sites associated with CCR5-specific ZFNs and TALENs reside in CCR2, a close homolog of CCR5, located 15 kbp upstream of CCR5. To avoid off-target mutations in CCR2 and unwanted chro-mosomal rearrangements, we intentionally chose the target site of our RGEN to recognize a region within the CCR5 sequence that has no apparent homology with the CCR2 sequence.

To investigate whether the CCR5-specific RGEN was associated with off-target effects, we searched for potential off-target sites in the human genome that are most homologous to the 23-bp target sequence. As expected, no such sites were found in CCR2. Instead, we found four sites elsewhere in the human genome, each of which carries 3-base mismatches to the on-target site (Fig. 2b). The T7EI assays showed that mutations were not induced at these sites (assay sensitivity, ~0.5%). Furthermore, we used PCR to detect the induc-tion of chromosomal deletions in cells separately transfected with plasmids encoding either the ZFN or the chimeric RNA specific to CCR5 and a plasmid encoding Cas9. As expected, because of the dif-ference in target sites, the ZFN induced deletions, whereas gRNA-Cas9 did not (Supplementary Fig. 3). Although we did not detect any off-target effects with the CCR5-specific RGEN in this study,

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a b

c

-3′-5′3′-

3′-

5′-

5′-Chimeric RNA

Cas9

CCR5Chr. 3

1 23

pUC ori

ApaLI

KanR

1.7 kbp

Target site5.4 kbp

Cas9:Chimeric RNA:

Target sequence:

Circular

Linearized

Nicked

LinearizedSupercoiled

5.4 kbp3.7 kbp

1.7 kbp

–––

+––

–+–

++–

––+

+–+

–++

+++

PCMV

PCMV

mRFP eGFPStop

Target site

Out-of-frame

In-frame

sgRNA Cas9-induced DSBsNHEJ-mediated frameshift mutations

mRFP+

eGFP–

mRFP eGFP mRFP+

eGFP+

Indel formation

105

105

104

104

103

103

102

102

Reporter only

eGFP

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104

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105104103102

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104

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Reporter + Cas9

105104103102

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mR

FP

Reporter + Cas9 +chimeric RNA

it is possible that RGENs in general can induce off-target muta-tions at sites that carry mismatches distal from the PAM sequence2. In addition, deep sequencing and whole-genome sequencing may reveal off-target mutations induced by Cas9-based endonucleases. Further studies are warranted to clarify this issue.

Next, we reprogrammed Cas9 by replacing the CCR5-specific chi-meric RNA with a newly synthesized RNA designed to target human C4BPB, which encodes the beta chain of the C4b-binding protein, a transcription factor. This enzyme induced mutations at the chro-mosomal target site in human K562 cells at high, dose-dependent frequencies that ranged from 5% to 33% in three independent exper-iments (Fig. 2c and Supplementary Fig. 2b). Sequencing analysis of PCR amplicons revealed that, out of four mutant sequences, two clones contained a single-base or two-base insertion precisely at the cleavage site, a pattern that was also observed at the CCR5 site. These results indicate that sgRNA:Cas9 complexes cleave chromosomal DNA at expected positions.

The RGENs used in this study induced no obvious cytotoxicity. Mutation frequencies were stably maintained even 8 d after trans-fection (Supplementary Fig. 4), which allowed us to isolate clonal populations of mutant cells by limiting dilution (Supplementary Fig. 5). As expected, the gRNA-Cas9 endonucleases produced ~1 double-strand break in each cell, which was detected by TP53BP1 staining (Supplementary Fig. 6).

ZFNs and TALENs enable targeted mutagenesis in a variety of eukaryotic cells, plants and animals, but the mutation frequen-cies obtained with individual nucleases vary widely. Furthermore, some ZFNs and TALENs fail to show any genome editing activi-ties12,13 and it is technically challenging and time consuming to make custom nucleases. In this regard, RGENs could provide use-ful options for genome editing. Compared to ZFNs and TALENs, RGENs can be more readily customized because, as only the RNA component has to be replaced to make a new genome-editing nuclease, no subcloning steps are involved to make Cas9-based nucleases with new specificities. (While this paper was under review,

two other groups independently reported genome editing in mammalian cells using the gRNA-Cas9 system, but their methods require a subcloning step to make plasmids that encode the gRNA14,15.) The relatively small size of the Cas9 gene (4.1 kbp) compared with a pair of TALEN genes (~6 kbp) provides an advantage for this system in gene delivery. These features will make RGENs scalable and con-venient tools for genome engineering in cells and organisms.

Unlike ZFNs and TALENs, whose specificities of DNA recognition are tunable by changing the number of DNA-binding modules, Cas9-based RGENs recognize target sequences of a fixed length. The spe-cificity of Cas9-based RGENs is further limited by the requirement for a 5′-GG-3′ dinucleotide in the PAM sequence. Thus, RGENs can be designed to cleave DNA once per 8 bp (= 4 × 4/2) on average. This limitation might be relieved by engineering Cas9. Whereas FokI-based ZFNs and TALENs produce 5′ overhangs at cleavage sites, Cas9-based RGENs yield blunt ends rather than cohesive ends2. Our results show that double-strand breaks with blunt ends can also be readily repaired in mammalian cells. It would be interesting to inves-tigate how and whether blunt, double-strand-break ends are differ-entially repaired by the endogenous end-joining processes.

Figure 1 RGEN-catalyzed cleavage of plasmid DNA in vitro and in cellula. (a) Schematic representation of target DNA and chimeric RNA sequences. Red triangles indicate cleavage sites. The PAM sequence recognized by Cas9 is shown in bold. The sequences in the chimeric RNA derived from crRNA and tracrRNA are shown in red and blue, respectively. (b) In vitro cleavage of plasmid DNA by Cas9. An intact circular plasmid or ApaLI-digested plasmid was incubated with Cas9 and chimeric RNA. (c) RGEN-induced mutagenesis at an episomal target site. (Top) Schematic overview of cell-based assays using a RFP-GFP reporter. (Bottom) Flow cytometry of cells transfected with Cas9 and chimeric RNA. The percentage of cells that express RFP and GFP is indicated. DBSs, double-strand breaks; NHEJ, nonhomologous end joining.

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Two independent studies in this issue report RGEN-mediated genome engineering in microbial organisms16 and in zebrafish17, demonstrating broad applications of this new technology.

Note: Supplementary information is available in the online version of the paper.

ACKnoWledgMentSThis work was supported by the National Research Foundation of Korea (2012-0001225) and ToolGen, Inc. We thank Jae Kyung Chon for bioinformatic analysis.

AUtHoR ContRIBUtIonSS.W.C., S.K. and J.M.K. performed the experiments. J.-S.K. wrote the manuscript.

CoMPetIng FInAnCIAl InteReStSThe authors declare competing financial interests: details are available in the online version of the paper.

Published online at http://www.nature.com/doifinder/10.1038/nbt.2507. reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. Nature 482, 331–338 (2012).2. Jinek, M. et al. Science 337, 816–821 (2012).3. Cohen, J. Science 332, 784–789 (2011).4. Kim, H., Um, E., Cho, S.R., Jung, C. & Kim, J.S. Nat. Methods 8, 941–943 (2011).5. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W. & Kim, J.S. Genome Res. 19, 1279–1288

(2009).6. Miller, J.C. et al. Nat. Biotechnol. 29, 143–148 (2011).7. Mussolino, C. et al. Nucleic Acids Res. 39, 9283–9293 (2011).8. Perez, E.E. et al. Nat. Biotechnol. 26, 808–816 (2008).9. Kim, E. et al. Genome Res. 22, 1327–1333 (2012).10. Lee, H.J., Kweon, J., Kim, E., Kim, S. & Kim, J.S. Genome Res. 22, 539–548

(2012).11. Lee, H.J., Kim, E. & Kim, J.S. Genome Res. 20, 81–89 (2010).12. Kim, J.S., Lee, H.J. & Carroll, D. Nat. Methods 7, 91, author reply 91–92 (2010).13. Reyon, D. et al. Nat. Biotechnol. 30, 460–465 (2012).14. Cong, L. et al. Science doi:10.1126/science.1231143 (3 January 2013).15. Mali, P. et al. Science doi:10.1126/science.1232033 (3 January 2013).16. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of

bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.2508 (29 January 2013).

17. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.2501 (29 January 2013).

a bCCR5

ADCY5KCNJ6CNTNAP2Chr. 5 N/A

c

8.3% (4/48) mutated (chimeric RNA, 20 µg)

WT+1+2

–180

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C4BPB

–

160 20 40

159.3 19 23

80 160– –

+Cas9 plasmid:

Indels (%):

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CCR5

Cas9 plasmid:

Indels (%):

–

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+

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3.5 7.0 13 13

40 80 160Chimeric RNA (µg):

7.3% (7/96) mutated (chimeric RNA, 40 µg)

WT+1

–13–14–18–19–24–30

ADCY5 KCNJ6RGEN – + – +

CNTNAP2 Chr. 5 N/ARGEN – + – +

Figure 2 RGEN-driven mutations at endogenous chromosomal sites. (a) CCR5 locus. (b) Undetectable off-target mutations. (Top) On-target and potential off-target sequences. Mismatched bases are shown in blue. (Bottom) The T7EI assay was used to investigate whether these sites were mutated by the sgRNA:Cas9. N/A, not applicable, an intergenic site. (c) C4BPB locus. (Top) The T7EI assay was used to detect sgRNA:Cas9 mutations. Arrow indicates the expected positions of DNA bands cleaved by T7EI. Mutation frequencies (indels (%)) were calculated by measuring the band intensities). (Bottom) DNA sequences of the CCR5 and C4BPB wild-type (WT) and mutant clones. The region of the target sequence complementary to the chimeric RNA is shown in red. The column on the right indicates the number of inserted or deleted bases.

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Journal : Nature Biotechnology

Supplementary Information

Targeted genome engineering in human cells with RNA-guided endonucleases

Seung Woo Cho†, Sojung Kim†, Jong Min Kim, and Jin-Soo Kim*

National Creative Research Initiatives Center for Genome Engineering and Department of

Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea

†These authors contributed equally to this work.

*Correspondence should be addressed to J.-S.K. (jskim01@snu.ac.kr). Table of Contents Supplementary Methods Supplementary Figure 1. Amino-acid sequence of the expressed version of Cas9 used

in this study. Supplementary Figure 2. Reproducible genome editing in human cells with RGENs. Supplementary Figure 3. DNA sequences of mutant clones. Supplementary Figure 4. CCR5-specific RGEN did not induce off-target-associated

chromosomal deletions.

Supplementary Figure 5. Stable maintenance of RGEN-induced mutant cells. Supplementary Figure 6. RGEN-induced DSBs in cells detected by TP53BP1 staining. Supplementary Table 1. Oligonucleotides used in this study.

2

Supplementary Methods Construction of Cas9-encoding plasmids. The Cas9-coding sequence (4,104 bp), derived

from Streptococcus pyogenes strain M1 GAS (NC_002737.1), was reconstituted using the

human codon usage table and synthesized using oligonucleotides. First, 1-kbp DNA

segments were assembled using overlapping ~35-mer oligonucleotides and Phusion

polymerase (New England Biolabs) and cloned into T-vector (SolGent). A full-length Cas9

sequence was assembled using four 1-kbp DNA segments by overlap PCR. The Cas9-

encoding DNA segment was subcloned into p3s, which was derived from pcDNA3.1

(Invitrogen). In this vector, the Cas9 protein is expressed under the control of the CMV

promoter and is fused to a peptide tag (NH2-GGSGPPKKKRKVYPYDVPDYA-COOH)

containing the HA epitope and a nuclear localization signal (NLS) at the C-terminus

(Supplementary Fig. 1). Expression and nuclear localization of the Cas9 protein in K562

cells were confirmed by western blotting using anti-HA antibody (Santa Cruz). In vitro DNA cleavage assay. The Cas9 cassette was subcloned into pET28-b(+) and

transformed into BL21(DE3). The expression of Cas9 was induced using 0.5 mM IPTG for 4

h at 25 ℃. The Cas9 protein containing the His6-tag at the C terminus was purified using Ni-

NTA agarose resin (Qiagen) and dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1

mM DTT, and 10% glycerol. Purified Cas9 (50 nM) was incubated with super-coiled or pre-

digested plasmid DNA (300 ng) and sgRNA (50 nM) in a reaction volume of 20 μl in NEB

buffer 3 for 1 h at 37 ℃. Digested DNA was analyzed by electrophoresis using 0.8% agarose

gels. RNA preparation. RNA was in vitro transcribed through run-off reactions by T7 RNA

polymerase using the MEGAshortscript T7 Kit (Ambion) according to the manufacturer’s

manual. Templates for RNA in vitro transcription were generated by annealing two

complementary oligonuceotides (Supplementary Table 1). Transcribed RNA was resolved

on a 8% denaturing urea-PAGE gel. The gel slice containing RNA was cut out and

transferred to elution buffer. RNA was recovered in nuclease-free water followed by

phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA

was quantified by spectrometry. Cell culture. HEK 293T/17 (ATCC, CRL-11268) cells were maintained in Dulbecco’s

modified Eagle’s medium (DMEM) supplemented with 100 units/mL penicillin, 100 μg/mL

3

streptomycin, 0.1 mM nonessential amino acids, and 10% fetal bovine serum (FBS). K562

(ATCC, CCL-243) cells were grown in RPMI-1640 with 10% FBS and the

penicillin/streptomycin mix (100 U/ml and 100 μg/ml, respectively). Genome-editing assay. To introduce DSBs in mammalian cells using RGENs, 2x106 K562

cells were transfected with 20 μg of Cas9-encoding plasmid using the 4D-Nucleofector, SF

Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer’s

protocol. After 24 h, 20 to 160 μg of in vitro transcribed sgRNA was transfected into 1x106

K562 cells. Cells were collected at various time points after RNA transfection and genomic

DNA was isolated. The region including the target site was PCR-amplified using the primers

described in Supplementary Table 1. The amplicons were subjected to the T7E1 assay as

described previously6. For sequencing analysis, PCR products corresponding to genomic

modifications were purified and cloned into the T-Blunt vector using the T-Blunt PCR Cloning

Kit (SolGent). Cloned products were sequenced using the M13 primer. Reporter construct. The RFP-GFP reporter plasmids used in this study were constructed

as described previously5. Oligonucleotides corresponding to target sites (Supplementary

Table 1) were synthesized (Macrogen) and annealed. The annealed oligonucleotides were

ligated into a reporter vector digested with EcoRI and BamHI. Episomal reporter assay. HEK 293T cells were co-transfected with Cas9-encoding plasmid

(0.8 μg) and the RFP-GFP reporter plasmid (0.2 μg) in a 24-well plate using Lipofectamine

2000 (Invitrogen). At 12h post transfection, sgRNA (1 μg) prepared by in vitro transcription

was transfected using Lipofectamine 2000. At 3 d post-transfection, transfected cells were

subjected to flow cytometry and cells expressing both RFP and GFP were counted.

4

Supplementary Figures Supplementary Figure 1. Amino-acid sequence of the expressed version of Cas9 used in this study. The amino-acid sequence of the Cas9 protein encoded in our Cas9

expression plasmid is presented. The sequence of the nuclear localization signal and the HA

epitope at the C-terminus is underlined.

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE 60 ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG 180 NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 240 VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN 300 LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 360 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 420 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 480 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE 540 VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 600 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 660 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG 720 RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 780 HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER 840 MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH 900 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 960 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS 1020 KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 1080 MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF 1140 ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 1200 YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 1260 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE 1320 QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 1380 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGPPKKKRKV 1440 YPYDVPDYA*

5

Supplementary Figure 2. Reproducible genome editing in human cells with RGENs. (a) CCR5 locus. (b) C4BPB locus. The results of three independent experiments were

shown. The average mutation frequencies were shown in Fig. 2.

6

Supplementary Figure 3. DNA sequences of mutant clones. RGEN-induced mutant clones were isolated by limiting dilution from populations of K562

cells transfected with the Cas9 plasmid and sgRNA (100 μg and 40 μg used for CCR5 and

C4BPB, respectively). The region of the target sequence complementary to the sgRNA is

shown in red. The PAM sequence recognized by Cas9 is shown in bold. The column on the

right indicates the number of inserted or deleted bases.

a. CCR5, 11% (= 5/46) mutated

wt …CAATCTATGACATCAATTATTATACATCGGAGCCCTGCCAAA… clone 1 …CAATCTATGACATCAATTATTATA------AGCCCTGCCAAA… -6 clone 2 …CAATCTATGACAT--------------CGGAGCCCTGCCAAA… -14 clone 3 …CAATCTATGACAT--------------CGGAGCCCTGCCAAA… -14 clone 4 …CAATCTATGACATCAATTATTATA------AGCCCTGCCAAA… -6 clone 5 …CAATCTATGACATCAATTATTATACAT//240bp//CGGAGCCCT… +240

b. C4BPB, 21% (= 7/33) mutated

wt …TATGTGCAATGACCACTACATCCTCAAGGGCAGCAATCGGAG…

clone 1 …TATGTGCAATGACCACTACATC------------AATCGGAG… -12

clone 2 …TATGTGCAATGA------------------------TCGGAG… -24

clone 3 …TATGTGCAATGACCACTACAT---------CAGCAATCGGAG… -9

clone 4 …TATGTGCAATGACCACTACATCC--------AGCAATCGGAG… -8

clone 5a …TATGTGCAATGACCACTACATCCT----------------GAG… -15 clone 5b …TATGTGCAATGACCACTACATCCTTCAAGGGCAGCAATCGGAG… +1

clone 6 …TATGTGCAATGACCACTACATCCTCCTCAAGGGCAGCAATCGGAG… +3

clone 7 …TATGTGCAATGACCACTA----//78bp//---CAATCGGAG… -15,+78

7

Supplementary Figure 4. CCR5–specific RGEN did not induce off-target-associated

chromosomal deletions. The CCR5-specific RGEN and ZFN were expressed in human

cells. PCR was used to detect the induction of the 15-kb chromosomal deletions in these

cells.

8

Supplementary Figure 5. Stable maintenance of RGEN-induced mutant cells. Cells transected with RGENs were cultured for several days. Mutation frequencies were

measured using the T7E1 assay at 3 d, 5 d, and 8 d post-transfection. 40 μg sgRNA was

used.

9

Supplementary Figure 6. RGEN-induced DSBs in cells detected by TP53BP1 staining.

HeLa cells transfected with the C4BPB-specific RGEN (160 μg sgRNA) were fixed on glass

slides and incubated first with anti-TP53BP1 rabbit polyclonal antibodies (Bethyl

Laboratories) and then with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen-

Molecular Probes). Cells were mounted in the presence of DAPI (Sigma) and examined

under an immunofluorescence microscope (Zeiss). DAPI (blue) and TP53BP1 (green)

images were merged. Etoposide (1 μM) was used as a positive control. The average

number of foci per cell and the standard error of the mean are shown at the bottom of each

picture.

10

Supplmentary Tables Supplementary Table 1. Oligonucleotides used in this study. Oligonucleotides used for the construction of the reporter plasmid Directiona Sequence (5' to 3')

CCR5 F AATTCATGACATCAATTATTATACATCGGAGGAG R GATCCTCCTCCGATGTATAATAATTGATGTCATG

Primers used in the T7E1 assay Gene Directiona Sequence (5' to 3')

CCR5

F1 CTCCATGGTGCTATAGAGCA F2 GAGCCAAGCTCTCCATCTAGT

R GCCCTGTCAAGAGTTGACAC

C4BPB

F1 TATTTGGCTGGTTGAAAGGG R1 AAAGTCATGAAATAAACACACCCA F2 CTGCATTGATATGGTAGTACCATG R2 GCTGTTCATTGCAATGGAATG

Primers used for the amplification of off-target sites Gene Directiona Sequence (5' to 3')

ADCY5

F1 GCTCCCACCTTAGTGCTCTG R1 GGTGGCAGGAACCTGTATGT F2 GTCATTGGCCAGAGATGTGGA R2 GTCCCATGACAGGCGTGTAT

KCNJ6 F GCCTGGCCAAGTTTCAGTTA

R1 TGGAGCCATTGGTTTGCATC R2 CCAGAACTAAGCCGTTTCTGAC

CNTNAP2 F1 ATCACCGACAACCAGTTTCC F2 TGCAGTGCAGACTCTTTCCA R AAGGACACAGGGCAACTGAA

N/A Chr. 5

F1 TGTGGAACGAGTGGTGACAG R1 GCTGGATTAGGAGGCAGGATTC F2 GTGCTGAGAACGCTTCATAGAG R2 GGACCAAACCACATTCTTCTCAC

Primers used for the detection of chromosomal deletions Directiona Sequence (5' to 3')

Deletion F CCACATCTCGTTCTCGGTTT R TCACAAGCCCACAGATATTT

11

In vitro transcription templates Directiona Sequence (5' to 3')b

CCR5

F GAAATTAATACGACTCACTATAGGTGACATCAATTATTATACATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

R CGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACATGTATAATAATTGATGTCACCTATAGTGAGTCGTATTAATTTC

C4BPB

F GAAATTAATACGACTCACTATAGGAATGACCACTACATCCTCAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

R CGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTGAGGA TGTAGTGGTCATTCCTATAGTGAGTCGTATTAATTTC

a F, forward; R, reverse b Sequences complementary to target DNA are shown in bold. The T7 promoter sequence is underlined.