Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells
- Authors
- Kim, Daesik; Kim, Jungeun; Hur, Jun ho; Been, Kyung Wook; Yoon, Sun-heui; Kim, Jin-Soo
- Issue Date
- Aug-2016
- Publisher
- NATURE PUBLISHING GROUP
- Citation
- NATURE BIOTECHNOLOGY, v.34, no.8, pp.863 - 868
- Indexed
- SCIE
SCOPUS
- Journal Title
- NATURE BIOTECHNOLOGY
- Volume
- 34
- Number
- 8
- Start Page
- 863
- End Page
- 868
- URI
- https://scholarworks.bwise.kr/hanyang/handle/2021.sw.hanyang/154122
- DOI
- 10.1038/nbt.3609
- ISSN
- 1087-0156
- 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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