Genome editing techniques have been rapidly developing in recent decades1. Among them, site-specific cleavage of genomic loci in various organisms by homing endonucleases (HEases)2, Zinc finger nucleases (ZFNs)3, transcription activator-like effector nucleases (TALENs)4, and most recently the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 system5, has been utilized widely not only in laboratories but also for translational studies. The central issue of genome editing is how to achieve specific and robust recognition of particular genomic sequences. In the case of HEases, ZFNs, and TALENs, this is achieved by specific intermolecular interactions between nucleotides and protein motifs, while for CRISPR/Cas9, the specificity is due to Watson-Crick base pairing between CRISPR RNA (crRNA) and its recognition sequences. The crRNA targets a 20-bp complementary target DNA sequence, which is flanked by a proto-spacer adjacent motif (PAM). Recent crystal structure studies and single-molecule DNA curtain experiments suggested that PAM site is essential for the initiation of Cas9 binding while the seed sequence corresponding to 3′ end of the crRNA complementary recognition sequence which is also directly adjacent to PAM is critical for subsequent Cas9 binding, R-loop formation, and activation of nuclease activities in Cas96,7,8. While extensive efforts have been focused on optimizing the efficiency of targeting and cleavage by the CRISPR/Cas9 system in various organisms, relatively few studies investigated the mistargeting, so called off-targeting, activities9,10,11,12. In those studies, limited numbers of potential target DNA sequences with point or combined mismatches in comparison with the authentic targeting sites were tested for in vitro and in vivo cleavage activities. While these studies suggest the off-targeting activities of Cas9 on those examined sites are limited, for a comprehensive view an unbiased genome-wide analysis is required. Here, by a robust unbiased approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro, suggesting that the off-target cleavage activity of CRISPR/Cas9 in human genome is very limited.

We performed ChIP-seq analysis in HEK-293T cells co-transfected which DM-Cas9-CV and no sgRNA or sgRNAs targeting either the emx1 or efga-t1 locus. Biological repeats were performed to reduce potential noises in the assay. In pooled ChIP-seq libraries, the original targeting sites of emx1, egfa-t1, as well as the known off-target of emx1, emx1-OT4, showed significant specific sgRNA-dependent enrichment (Figure 1D). To achieve more stringent identification of the off-targets, during MACS peak calling, we set the threshold as FDR < 0.5%. In ChIP-seq libraries generated from 293T cells without transfected sgRNA, no peak was identified, while in libraries generated from cells transfected with egfa-t1 sgRNA, only the original target site was identified in biological repeats (Supplementary information, Table S1A). For emx1 sgRNA, 50 and 63 peaks were identified in each biological repeat, and 12 overlapped peaks were finally obtained (Figure 1E). Interestingly, most of the 50 (39/50) and 63 (42/63) peaks contain conserved motifs which correlate well with PAM and its 5′ 10-12 bp seed region, while all 12 peaks that appeared in both biological repeats contain such conserved motifs (Figure 1F and Supplementary information, Figure S1J-S1L and Table S1C-S1E). We further confirmed these identified peaks by quantitative PCR. Most of them showed significant sgRNA-dependent specific enrichment, with some showing comparable enrichment as the original emx1 locus (Figure 1G and Supplementary information, Figure S1M). Finally, we checked whether these sites corresponding to peaks could indeed be cut by Cas9. Surprisingly, in both in vitro and in vivo cleavage assays, most of these binding off-targets could not be significantly cleaved while the emx1 original site and its known off-target (emx1-OT4) were almost completely cleaved by Cas9/sgRNAemx1 (Figure 1H and Supplementary information, Figure S1I, S1N). Only two binding off-targets, OT2-1 and OT2-4, reproducibly showed weak cleavage (Figure 1H and Supplementary information, Figure S1N). These results suggest that substrate binding could be uncoupled from the cleavage step in the CRISPR/Cas9 system.

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