RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry

Area of Science:

• Molecular Biology
• Microbial Genetics
• Structural Biology

Background:

• Bacterial phages use anti-CRISPR (ACR) factors to evade CRISPR-Cas adaptive immunity.
• While most ACRs are proteins, RNA ACRs (rAcrs) have emerged, mimicking CRISPR RNAs (crRNAs) to inhibit Cas nucleases.
• Understanding diverse ACR mechanisms is crucial for harnessing CRISPR-Cas systems.

Purpose of the Study:

• To discover and characterize novel RNA anti-CRISPRs (rAcrs) targeting CRISPR-Cas systems.
• To investigate the mechanism of inhibition employed by a newly identified plasmid-encoded rAcr in *Listeria seeligeri*.
• To elucidate the structural basis of RNA-based immune evasion in bacteria.

Main Methods:

• Identification and cloning of a plasmid-encoded small RNA, rAcrVIA1, from *Listeria seeligeri*.
• Biochemical assays to confirm the inhibitory activity of rAcrVIA1 against the CRISPR-Cas13 system.
• Cryo-electron microscopy (cryo-EM) to determine the structure of the Cas13-rAcrVIA1 complex.

Main Results:

• Discovery of rAcrVIA1, a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system.
• Structural analysis revealed that rAcrVIA1 adopts a fold highly similar to crRNA.
• Inhibition by rAcrVIA1 occurs despite minimal sequence homology to crRNA, indicating structural mimicry.

Conclusions:

• The findings expand the known repertoire of RNA anti-CRISPRs (rAcrs).
• rAcrVIA1 provides a novel example of immune antagonism mediated by RNA structural mimicry.
• This discovery highlights the diverse strategies bacteria and phages employ in their molecular arms race.