Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5′ ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
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This research was supported by grants from the Institute for Basic Science (IBS-R021-D1) to J.-S.K., Korea Health Industry Development Institute (HI14C1277) to J.-I.K., and Korea Institute of Planning and Evaluation for Technology of Food, Agriculture, Forestry and Fisheries (311011-05-3-SB010) to S.K.
E.K., H.R.Y. and S.K. are employees of ToolGen, Inc. J.-S.K. is a shareholder of ToolGen, Inc.
Integrated supplementary information
(a) T7E1 assay confirming gene knockout in clonal populations of HAP1 haploid cells. WT, wild-type; MT, mutant; WT+MT, a 1:1 mixture of WT and MT PCR amplicons. (b) DNA sequences of wild-type and mutant clones. The PAM is shown in blue. Inserted bases are shown in red.
(a) Small deletion in the ERBB3 KO clone confirmed by Sanger sequencing. (b) RGEN-mediated mutagenesis at the on-target and potential off-target sites. Mutation frequencies (%) were measured using T7E1 and targeted deep sequencing. (c) The on-target mutant sequence in the FGFR4 KO clone. The PAM sequence is shown in blue and inserted bases are shown in red. (d) Integrative Genomics Viewer (IGV) image at the FGFR4 on-target site.
(a) The number of potential off-target sites that differ from on-target sites by up to 8 nucleotides or by 2 nucleotides with a DNA or RNA bulge of up to 5 nucleotides in length. (b) Schematic of consensus sequence generation. (c) On-target mutations in five KO clones identified by consensus sequence comparison.
(a-d) Representative IGV images obtained using the HBB-specific RGEN at the potential off-target sites OT1 (a), OT3 (b), OT7 (c), and OT12 (d). An indel is indicated by an arrow (a) or shown in a box (b).
(a) An IGV image at a nuclease cleavage site. (b, c) 5’ End plots showing the absolute and relative number of sequence reads with the same 5’ end across nucleotide positions at the OT1 (b) and OT3 (c) sites.
(a-c) Representative IGV images around false-positive sites that resulted from naturally-occurring indels in HAP1 cells.
(a, b) Off-target indels were detected by targeted deep sequencing. Inserted nucleotides are shown in red and the PAM sequence is shown in blue.
(a) 5’ End plots at the one of VEGF-A off-target site. (b) Heatmap comparing digenome-captured sites with the on-target site. Dark red and dark blue correspond to 100% and 0% matches, respectively, at a given position. (c) Sequence logo obtained via WebLogo using DNA sequences at digenome-captured sites. (d) Summary of Digenome-seq and targeted deep sequencing. N.D., not determined. (e) Off-target sites validated by targeted deep sequencing. Blue and red bars represent indel frequencies obtained using mock-transfected HAP1 cells and the VEGF-A RGEN-transfected HAP1 cells, respectively. (Left) DNA sequences of on-target and off-target sites. Mismatched bases are shown in red. The PAM is shown in blue. (Right) P value was calculated by the Fisher exact test. Additional deep sequencing results can be found in Supplementary Table 3.
Supplementary Figure 9 RGEN-induced digenome sequencing to capture off-target sites of the VEGFA-targeting RGEN.
(a-d) 5’ End plots showing the absolute and relative number of sequence reads with the same 5’ end across nucleotide positions in on-target (a) and off-target region (b-d).
Supplementary Figure 10 Indel sequences induced by the VEGFA RGEN at newly validated off-target sites.
(a-d) Off-target indels were detected by targeted deep sequencing. Inserted nucleotides are shown in red and the PAM sequence is shown in blue.
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Kim, D., Bae, S., Park, J. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12, 237–243 (2015). https://doi.org/10.1038/nmeth.3284
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