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. 2016 Aug;34(8):863-8.
doi: 10.1038/nbt.3609. Epub 2016 Jun 6.

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

Daesik Kim  1   2 , Jungeun Kim  1   2 , Junho K Hur  1 , Kyung Wook Been  1   2 , Sun-Heui Yoon  2 , Jin-Soo Kim  1   2
Affiliations

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

Daesik Kim et al. Nat Biotechnol. 2016 Aug.

Erratum in

Abstract

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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