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Cited by 4CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

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Structural basis of a dual-function type II-B CRISPR–Cas9

Open access
Hibshman, Grace N, Taylor, David W·2025·Nucleic Acids Research
DOI 10.1093/nar/gkaf585PMC PMC12231574

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Abstract

Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA–RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature—the REC3 clamp—that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR–Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.

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Metadata

Authors
Hibshman, G. N., & Taylor, D. W.
Affiliations
The University of Texas at Austin (US)
Corresponding author
Taylor, David W
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2025-06-20
Volume
53
Issue
12
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkaf585
PMC
PMC12231574
OpenAlex
W4412034271
Cited by
4
References
6
Citations by year
2026: 2 · 2025: 2
FWCI
1.20
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Institutes of Health (R35GM138348)
  • Welch Foundation (F-1938)
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
Francisella novicida, off-target editing, R-loops, transcriptional repression, lipoprotein gene, host immune evasion, antibacterial strategies, genome editing specificity

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