Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells
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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AsCas12a has different PAM requirements and natural very high specificity.
“Among these, AsCas12a has been widely used for its different PAM requirements and a natural very high specificity17–20.”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [20]
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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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