Cited by 102CRISPR and Genetic EngineeringRNA regulation and diseaseEnzyme Structure and Function

Structural basis for the altered PAM recognition by engineered CRISPR-Cpf1

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Nishimasu, Hiroshi, Yamano, Takashi, Gao, Linyi, Zhang, Feng, Ishitani, Ryuichiro, Nureki, Osamu·2017·Molecular Cell

Abstract

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.

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Crossref · 10.1016/j.molcel.2017.04.019

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  • Due to recognition preference of some specific bases of CRISPR/Cas9 system and diverse epigenetic landscape, different targeted gene loci may exhibit distinctive different gene editing efficiencies.

    “Besides, due to the recognition preference of some specific bases of CRISPR/Cas9 system and the diverse epigenetic landscape, different targeted gene loci may exhibit distinctive different gene editing efficiencies [140, 201–203].”

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Authors
Nishimasu, H., Yamano, T., Gao, L., Zhang, F., Ishitani, R., & Nureki, O.
Affiliations
  • Japan Science and Technology Agency (JP)
  • The University of Tokyo (JP)
  • Broad Institute (US)
Corresponding author
  • Nishimasu, Hiroshi
  • Nureki, Osamu
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2017-07
Volume
67
Issue
1
Pages
139-147.e2
ISSN
1097-2765 (ISSN-L)
OpenAlex
W2622596320
Cited by
102
References
36
Citations by year
2026: 4 · 2025: 12 · 2024: 10 · 2023: 9 · 2022: 14 · 2021: 11 · 2020: 9 · 2019: 15 · 2018: 12 · 2017: 6
FWCI
4.69
Type
journal article
Language
en
Open access
Locations
  • Molecular Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
Funders
  • JST
  • PRESTO (JPMJPR13L8)
  • JSPS KAKENHI (26291010, 15H01463)
  • NIH
  • National Institute of Mental Health (5DP1-MH100706, 1R01-MH110049)
  • NSF
  • Howard Hughes Medical Institute
  • New York Stem Cell
  • Simons
  • Paul G. Allen Family
  • Vallee Foundations
  • James and Patricia Poitras
  • Robert Metcalfe
  • David Cheng
  • Japan Agency for Medical Research and Development
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
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
Keywords
CRISPR/Cas12a, protospacer adjacent motif, PAM recognition, crystal structure, genome editing, DNA cleavage
MeSH terms
Gene Editing, Bacterial Proteins, DNA, Bacterial, Escherichia coli, Models, Molecular, Mutation, Nucleic Acid Conformation, Nucleic Acid Heteroduplexes, Protein Binding, Protein Conformation, RNA, Structure-Activity Relationship, Acidaminococcus, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins

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