Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3′ PAM-distal region, but not in the 5′ PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3′ PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.
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This work was supported by IBS-R021-D1. We thank F. Zhang (MIT) for providing human codon-optimized Cpf1 expression plasmids.
D.K., J.K., J.K.H. and J.-S.K. have filed a patent application based on this work.
Integrated supplementary information
Indel frequencies obtained with mismatched crRNAs were measured by targeted deep sequencing. Error bars represent s.e.m. (n = 3). Some mismatched crRNAs appeared more active than the perfectly-matched crRNA but the differences were not statistically significant (Student's t-test, P > 0.4).
In vitro cleavage sites were determined via mononoplex Digenome-seq with LbCpf1 (n = 8), AsCpf1 (n = 8), and SpCas9 (n = 2) in this study and by multiplex Digenome-seq with SpCas9 (n = 11) in our previous study11.
Sequence logos of Digenome-captured sites obtained using AsCpf1 (Left) and LbCpf1 (Right). The number below sequence logos, n, indicates the number of Digenome-captured sites.
Supplementary Figure 5 Poor correlation between the number of Digenome-captured sites and the number of homologous sites with 6 or fewer mismatches in the human genome.
Sequence logos were obtained via WebLogo using Digenome-captured sites. Note that just the single DNMT1-4 on-target site was captured with LbCpf1 and AsCpf1.
Supplementary Figure 7 Poor correlations between indel frequencies at on-target sites and genome-wide target specificities of Cpf1 and Cas9.
SpCas9 indel frequencies at on-target sites and numbers of Digenome-seq positive sites were from our previous study11.
Indel frequencies at Digenome-captured sites were determined via targeted deep sequencing in HEK293T17 cells transfected with AsCpf1 (red) or LbCpf1 (blue) plasmids.
Supplementary Figure 9 Indel frequencies at on-target and off-target sites obtained with truncated and full-length crRNAs.
crRNAs truncated at the 3′ end (tru-crRNAs) were designed to match the DNMT1-3 target site. Each tru-crRNA was transfected with the AsCpf1 expression plasmid into HEK293T cells using lipofectamine 2000. After 72 hr, genomic DNA was isolated and indel frequencies at the on-target and off-target sites were measured by targeted deep sequencing. Error bars represent mean ± s.e.m.
(a) The number of mutant sequence reads binned by the deletion/insertion size in base pairs. Mutation signatures at the EMX1-2 site were obtained in HEK293T cells transfected with LbCpf1, AsCpf1, or SpCas9 plasmids using targeted deep sequencing. (b) Mutant sequences induced at the EMX1-2 target site. The most frequently-identified sequences are shown. PAM sequences are shown in blue. crRNA/sgRNA target sites are shown in red. Microhomology sequences are underlined and in blue. The number of deleted or inserted bases are shown on the right.
Full-length gel images of Fig. 1a. Unrelated lanes are indicated by cross.
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Kim, D., Kim, J., Hur, J. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34, 863–868 (2016). https://doi.org/10.1038/nbt.3609
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