Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition

Area of Science:

• Molecular Biology
• Structural Biology
• Microbial Genetics

Background:

• Bacteriophages encode anti-CRISPR proteins to counteract host CRISPR-Cas adaptive immunity.
• Type II-C Cas9 enzymes, including Nme1Cas9 and SmuCas9, are key components of CRISPR-Cas systems.
• AcrIIC5 is an anti-CRISPR protein known to inhibit specific type II-C Cas9 orthologs.

Purpose of the Study:

• To determine the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA.
• To elucidate the mechanism by which AcrIIC5 inhibits Cas9 activity.
• To investigate the evolutionary convergence of DNA-mimicking anti-CRISPR proteins.

Main Methods:

• X-ray crystallography to solve the structure of the AcrIIC5-Nme1Cas9-sgRNA complex.
• Biochemical assays to assess Cas9 inhibition.
• Site-directed mutagenesis to identify key interacting residues.

Main Results:

• The structure reveals AcrIIC5 adopts a novel fold that mimics DNA's charge and size.
• AcrIIC5 binds to the Cas9 protospacer adjacent motif (PAM) binding site, occluding DNA access.
• Key interactions involve AcrIIC5 binding in a crevice of Cas9, including the WED and PI domains.
• Mutational analyses identified critical residues on both AcrIIC5 and Cas9 for inhibition.

Conclusions:

• AcrIIC5 inhibits type II-C Cas9 by acting as a DNA mimic, binding the PAM site.
• The findings demonstrate convergent evolution among anti-CRISPR proteins employing DNA-mimicry.
• AcrIIC5 represents a potent inhibitor of type II-C Cas9 orthologs, offering potential biotechnological applications.