AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation

Area of Science:

• Molecular Biology
• Structural Biology
• Microbial Genetics

Background:

• Anti-CRISPR (Acr) proteins are phage-encoded inhibitors of CRISPR-Cas adaptive immunity.
• AcrIIC4 is a broad-spectrum inhibitor of type II-C CRISPR-Cas9 systems with an uncharacterized mechanism.
• Understanding Acr mechanisms is crucial for deciphering microbial defense systems and developing biotechnological tools.

Purpose of the Study:

• To elucidate the mechanism of inhibition of Haemophilus parainfluenzae Cas9 (HpaCas9) by AcrIIC4.
• To determine the structural basis for AcrIIC4's interaction with the HpaCas9-sgRNA-DNA complex.
• To reveal how AcrIIC4 prevents Cas9 nuclease activity.

Main Methods:

• X-ray crystallography was used to determine the structures of AcrIIC4 alone and in complex with HpaCas9-sgRNA and target DNA.
• Biochemical assays were performed to assess the functional impact of AcrIIC4 binding on Cas9 activity.

Main Results:

• AcrIIC4 binds to the crevice between the REC1 and REC2 domains of HpaCas9.
• AcrIIC4 binding restricts REC2 domain mobility, preventing target double-stranded DNA unwinding.
• The formation of a full-length guide RNA:target DNA heteroduplex and subsequent R-loop is blocked, inhibiting Cas9 nuclease activation.

Conclusions:

• AcrIIC4 employs a unique mechanism to inhibit Cas9 by preventing R-loop formation, a critical step for DNA cleavage.
• This inhibition occurs despite allowing initial DNA binding to the Cas9 effector complex.
• The findings provide structural insights into a novel anti-CRISPR strategy and its implications for CRISPR-Cas immunity.