AcrVA3 Is a Double Strand DNA-Cleaving Anti-CRISPR That Indirectly Inhibits Cas12

Area of Science:

• Molecular Biology
• Bacteriology
• Genetics

Background:

• CRISPR-Cas12 systems provide bacterial defense against foreign DNA.
• Bacteriophages evolve anti-CRISPR (Acr) proteins to counteract CRISPR immunity.
• Eight AcrV proteins targeting Cas12 are known, but AcrVA3's mechanism remained uncharacterized.

Purpose of the Study:

• To determine the high-resolution structure of AcrVA3.
• To investigate the inhibitory mechanism of AcrVA3 against CRISPR-Cas12 systems in vitro.
• To understand how AcrVA3's unique strategy contributes to phage survival.

Main Methods:

• High-resolution structural determination of AcrVA3.
• Biochemical assays to assess AcrVA3's inhibitory function on Cas12.
• In vitro cleavage assays to analyze AcrVA3's enzymatic activity.

Main Results:

• The high-resolution structure of AcrVA3 was determined.
• AcrVA3 was found to not directly inhibit Cas12.
• AcrVA3 demonstrated significant double-stranded DNA (dsDNA) cleavage activity.

Conclusions:

• AcrVA3 employs an indirect CRISPR-Cas12 inhibition mechanism via DNA degradation.
• This DNA-centric strategy is distinct from previously characterized Acr proteins.
• The findings expand the understanding of anti-CRISPR mechanisms and phage-host interactions.