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

Grace N Hibshman1,2, David W Taylor1,2,3,4

  • 1Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, United States.

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Cas9 from Francisella novicida (FnCas9) uses a REC3 clamp for high-fidelity DNA editing, unlike Streptococcus pyogenes Cas9 (SpCas9). This clamp enables precise genome editing and offers new antibacterial strategies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Streptococcus pyogenes Cas9 (SpCas9) is a revolutionary genome editing tool but can cause off-target edits due to DNA-RNA mismatch tolerance.
  • High-fidelity Cas9 variants are needed for precise genome engineering and therapeutic applications.

Purpose of the Study:

  • To investigate the structural basis of high-fidelity DNA targeting by Cas9 from Francisella novicida (FnCas9).
  • To explore the function of the unique REC3 clamp in FnCas9 and its potential applications.

Main Methods:

  • Kinetic and structural analyses of FnCas9.
  • Biochemical assays to determine DNA-RNA binding and cleavage activity.
  • Structural determination of FnCas9 in complex with scaRNA.

Main Results:

  • FnCas9 possesses a REC3 clamp, a unique structural feature responsible for its high-fidelity DNA targeting.
  • The REC3 clamp interacts with the R-loop, creating a checkpoint that enhances specificity.
  • FnCas9, guided by scaRNA, can repress gene expression, suggesting a role in subverting host immunity.
  • Structural data reveals how partial R-loop complementarity prevents cleavage and favors transcriptional repression.

Conclusions:

  • The REC3 clamp is a key determinant of FnCas9's high specificity, offering a potential avenue for engineering more precise genome editors.
  • FnCas9's ability to repress gene transcription via scaRNA presents novel antibacterial strategies.
  • The conserved nature of the REC3 clamp across type II-B CRISPR-Cas9 systems highlights its broad biotechnological and therapeutic potential.