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Cited by 24CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

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Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications

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Hu, Chunyi, Myers, Mason T., Zhou, Xufei, Hou, Zhonggang, Lozen, Macy L., Nam, Ki Hyun, Zhang, Yan, Ke, Ailong·2024·Molecular Cell
DOI 10.1016/j.molcel.2023.12.034PMC PMC10857747

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Abstract

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

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Metadata

Authors
Hu, C., Myers, M. T., Zhou, X., Hou, Z., Lozen, M. L., Nam, K. H., Zhang, Y., & Ke, A.
Affiliations
  • National University of Singapore (SG)
  • University of Michigan (US)
  • Kookmin University (KR)
  • Cornell University (US)
Corresponding author
  • Zhang, Yan
  • Ke, Ailong
ORCID
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2024-02
Volume
84
Issue
3
Pages
463-475.e5
ISSN
1097-2765 (ISSN-L)
DOI
10.1016/j.molcel.2023.12.034
PMC
PMC10857747
OpenAlex
W4391056248
Cited by
24
References
1
Citations by year
2026: 9 · 2025: 5 · 2024: 9
FWCI
3.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Science Foundation (1719875)
  • U.S. Department of Energy (KP1607011)
  • University of Michigan
  • National Institutes of Health (DMR-1719875, NIH T32)
  • Cornell Center for Materials Research (DMR‐1719875, 1719875)
  • National Institute of General Medical Sciences (T32GM007544, R35GM137883, R35GM118174)
  • Division of Materials Research (DMR-1719875, 1719875)
  • Biological and Environmental Research (KP1607011)
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 › Genetics › Bacterial Genetics and Biotechnology
Keywords
Cascade complex, anti-CRISPR proteins, PAM recognition, allosteric inhibition, genome editing, Neisseria lactamica, cryo-EM structure, transcriptional regulation

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