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PreprintCited by 26CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyDNA and Nucleic Acid Chemistry

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Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition

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Sun, Wei, Zhao, Xiaolong, Wang, Jinlong, Yang, Xiaoqi, Cheng, Zhi, Liu, Shuo, Wang, Jiuyu, Sheng, Gang, Wang, Yanli·2023·Nucleic Acids Research
DOI 10.1101/2023.01.17.524369PMC PMC9976890

Preprint — published as 10.1093/nar/gkad052

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Abstract

Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH structural domains. We employ biochemical and mutational analyses to build a model for AcrIIC5’s mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.

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Metadata

Authors
Sun, W., Zhao, X., Wang, J., Yang, X., Cheng, Z., Liu, S., Wang, J., Sheng, G., & Wang, Y.
Affiliations
Chinese Academy of Sciences (CN)
Corresponding author
  • Sun, Wei
  • Wang, Yanli
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2023-01-17
Volume
51
Issue
4
Pages
1984-1995
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2023.01.17.524369
PMC
PMC9976890
OpenAlex
W4319293517
Cited by
26
References
1
Citations by year
2026: 9 · 2025: 9 · 2024: 5 · 2023: 3
FWCI
2.32
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
Funders
  • National Natural Science Foundation of China (31930065, 32071444, 22121003, 91440201, 31630015, 32071198, 31725008, 91940302)
  • Chinese Academy of Sciences (QYZDY-SSW-SMC021, XDB37010202)
  • Youth Innovation Promotion Association of the Chinese Academy of Sciences (2021090)
  • National Key Research and Development Program of China (2017YFA0504203)
  • Youth Innovation Promotion Association
  • Institute of Biophysics, Chinese Academy of Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
Keywords
anti-CRISPR proteins, protein-DNA complex structure, convergent evolution, phage-host interactions
MeSH terms
CRISPR-Associated Protein 9, Binding Sites, DNA, Neisseria meningitidis, Neisseriaceae, Viral Proteins, CRISPR-Cas Systems

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