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. 1
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 (NH 2 -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 His 6 -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 predigested 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 2
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, 2x10 6 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 1x10 6 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 previously 6. 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 previously 5. 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. 3
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* 4
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. 5
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 6
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. 7
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. 8
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. 9
Supplmentary Tables Supplementary Table 1. Oligonucleotides used in this study. Oligonucleotides used for the construction of the reporter plasmid Direction a Sequence (5' to 3') F AATTCATGACATCAATTATTATACATCGGAGGAG CCR5 R GATCCTCCTCCGATGTATAATAATTGATGTCATG Primers used in the T7E1 assay Gene Direction a Sequence (5' to 3') F1 CTCCATGGTGCTATAGAGCA CCR5 F2 GAGCCAAGCTCTCCATCTAGT R GCCCTGTCAAGAGTTGACAC C4BPB F1 R1 F2 R2 TATTTGGCTGGTTGAAAGGG AAAGTCATGAAATAAACACACCCA CTGCATTGATATGGTAGTACCATG GCTGTTCATTGCAATGGAATG Primers used for the amplification of off-target sites Gene Direction a Sequence (5' to 3') F1 GCTCCCACCTTAGTGCTCTG ADCY5 R1 GGTGGCAGGAACCTGTATGT F2 GTCATTGGCCAGAGATGTGGA R2 GTCCCATGACAGGCGTGTAT F GCCTGGCCAAGTTTCAGTTA KCNJ6 R1 TGGAGCCATTGGTTTGCATC R2 CCAGAACTAAGCCGTTTCTGAC F1 ATCACCGACAACCAGTTTCC CNTNAP2 F2 TGCAGTGCAGACTCTTTCCA R AAGGACACAGGGCAACTGAA F1 TGTGGAACGAGTGGTGACAG N/A Chr. 5 R1 GCTGGATTAGGAGGCAGGATTC F2 GTGCTGAGAACGCTTCATAGAG R2 GGACCAAACCACATTCTTCTCAC Primers used for the detection of chromosomal deletions Direction a Sequence (5' to 3') F CCACATCTCGTTCTCGGTTT Deletion R TCACAAGCCCACAGATATTT 10
In vitro transcription templates Direction a Sequence (5' to 3') b CCR5 C4BPB F R F R GAAATTAATACGACTCACTATAGGTGACATCAATTATTATACATGTTTT AGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG CGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACATGTATA ATAATTGATGTCACCTATAGTGAGTCGTATTAATTTC GAAATTAATACGACTCACTATAGGAATGACCACTACATCCTCAAGTTT TAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG CGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTGAGGA TGTAGTGGTCATTCCTATAGTGAGTCGTATTAATTTC a F, forward; R, reverse b Sequences complementary to target DNA are shown in bold. The T7 promoter sequence is underlined. 11