Cited by 731CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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

Kim, Daesik, Kim, Jungeun, Hur, Junho K, Been, Kyung Wook, Yoon, Sun-heui, Kim, Jin-Soo·2016·Nature Biotechnology

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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Crossref · 10.1038/nbt.3609

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Authors
Kim, D., Kim, J., Hur, J. K., Been, K. W., Yoon, S.-H., & Kim, J.-S.
Affiliations
  • Seoul National University (KR)
  • Institute for Basic Science (KR)
Corresponding author
Kim, Jin-Soo
Venue
Nature Biotechnology
Published
2016-06-06
Volume
34
Issue
8
Pages
863-868
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
OpenAlex
W2410526873
Cited by
731
References
12
Citations by year
2026: 31 · 2025: 56 · 2024: 56 · 2023: 62 · 2022: 70 · 2021: 92 · 2020: 92 · 2019: 86 · 2018: 77 · 2017: 85 · 2016: 20
FWCI
46.41
Type
journal article
Language
en
Update notices
erratum (2016-08-09)
Open access
closed
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
CRISPR gene editing, targeting specificity, protospacer adjacent motif, PAM sequence, mismatch tolerance, off-target cleavage, ribonucleoprotein, human genome
MeSH terms
Bacterial Proteins, Binding Sites, Chromosome Mapping, Endonucleases, Enzyme Activation, Humans, Protein Binding, Substrate Specificity, Genome, Human, Base Pair Mismatch, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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