Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells

  • An Erratum to this article was published on 09 August 2016


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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Figure 1: Genome editing in human cells using Cpf1.
Figure 2: Specificity of Cpf1.
Figure 3: Using Digenome-seq to analyze genome-wide specificity of Cpf1 and SpCas9 RGENs.
Figure 4: Overhang patterns and mutation signatures of Cpf1 and SpCas9 RGENs.

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Change history

  • 18 July 2016

    In the version of this article initially published, the year in the received date on the first page was given as “2015,” but should be “2016.” The error has been corrected for the print, PDF and HTML versions of this article.


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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.

Author information




J.-S.K. supervised the research. D.K., J.K., J.K.H., K.W.B. and S.Y. carried out the experiments. D.K. performed bioinformatics analyses.

Corresponding author

Correspondence to Jin-Soo Kim.

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Competing interests

D.K., J.K., J.K.H. and J.-S.K. have filed a patent application based on this work.

Integrated supplementary information

Supplementary Figure 1 crRNA sequences.

Supplementary Figure 2 Specificity of Cpf1 examined using mismatched crRNAs at the AAVS1 sites.

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).

Supplementary Figure 3 The number of Digenome-seq captured sites with LbCpf1, AsCpf1, and SpCas9.

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.

Supplementary Figure 4 Sequence logos of Cpf1-mediated Digenome-captured sites.

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.

Supplementary Figure 6 Sequence logos of Digenome-captured sites.

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.

Supplementary Figure 8 Indel frequencies at Digenome-captured sites.

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.

Supplementary Figure 10 Mutation signatures of LbCpf1, AsCpf1, and SpCas9.

(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.

Supplementary Figure 11 Full-length gel images

Full-length gel images of Fig. 1a. Unrelated lanes are indicated by cross.

Supplementary Figure 12 In vitro DNA cleavage scoring system for Cpf1 Digenome-seq analysis.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Table 1 (PDF 1810 kb)

Supplementary Table 2

Indel frequencies at on-target and potential off-target sites. (PDF 366 kb)

Supplementary Table 3

Digenome-captured sites. (PDF 535 kb)

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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).

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