Structural basis for the altered PAM recognition by engineered CRISPR-Cpf1
Open accessAbstract
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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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].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [201]
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A highly adaptable platform powered by CRISPR-Cas12a to diagnose lumpy skin disease in cattle
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Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits
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Recent advances in CRISPR technologies for genome editing
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CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity
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Split CRISPR/Cas systems: Pioneering solutions for molecular diagnostics challenges
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PAM-free diagnostics with diverse type V CRISPR-Cas systemsPreprint
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CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics
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AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach
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Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E. coli
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Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing
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Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications
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Utilization of nicking properties of CRISPR-Cas12a effector for genome editing
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The applications of CRISPR/Cas-mediated genome editing in genetic hearing loss
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Protein Engineering of DNA-Dependent Enzymes
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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)
- PMC
- PMC5957533
- 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
- bronze
- doi.org
- Licence
- 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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