Structural snapshots of R-loop formation by a type I-C CRISPR Cascade

Area of Science:

• Molecular Biology
• Structural Biology
• Microbiology

Background:

• Type I CRISPR-Cas systems provide adaptive immunity against foreign nucleic acids.
• The Cascade effector complex is central to Type I CRISPR-Cas DNA targeting and cleavage.
• Understanding Cascade's mechanism is crucial for harnessing CRISPR-Cas for biotechnology.

Purpose of the Study:

• To elucidate the structural mechanisms of DNA target binding and R-loop formation by the Type I-C Cascade complex from *D. vulgaris*.
• To investigate the structural basis of anti-CRISPR (Acr) protein inhibition of Type I-C Cascade.
• To provide insights into the allosteric activation of Cascade upon target recognition.

Main Methods:

• X-ray crystallography was used to determine the structures of *D. vulgaris* Type I-C Cascade at various stages of DNA binding.
• Structural analysis was performed to understand protein-DNA interactions and allosteric conformational changes.
• Comparative structural analysis was employed to understand the inhibition mechanisms of two distinct anti-CRISPR proteins.

Main Results:

• Structures reveal a "molecular seatbelt" mechanism involving Cascade's belly subunits that stabilizes the non-target DNA strand.
• This stabilization facilitates directional R-loop formation and prevents dsDNA reannealing.
• Two anti-CRISPR proteins were shown to inhibit Cascade by distinct strategies that converge on blocking PAM scanning.

Conclusions:

• The study provides a detailed structural understanding of DNA recognition and R-loop formation by Type I-C Cascade.
• The findings reveal conserved and distinct mechanisms of inhibition employed by anti-CRISPR proteins.
• This work offers a structural foundation for understanding CRISPR-Cas immunity and its regulation.