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

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Title
Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells
Author(s)
Daesik Kim; Jungeun Kim; Junho K Hur; Kyung Wook Been; Sun-Heui Yoon; Jin-Soo Kim
Publication Date
2016-08
Journal
NATURE BIOTECHNOLOGY, v.34, no.8, pp.863 - 868
Publisher
NATURE PUBLISHING GROUP
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. © 2016 Nature America, Inc. All rights reserved
URI
https://pr.ibs.re.kr/handle/8788114/2800
ISSN
1087-0156
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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