Investigating the molecular mechanisms underlying the anti‐ CRISPR function of AcrIIA13b protein

Oct 25, 2025The FEBS journal

Molecular mechanisms behind the CRISPR-blocking function of the AcrIIA13b protein

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Abstract

The structure of AcrIIA13b from Staphylococcus haemolyticus reveals its role in inhibiting Cas9 activity.

AcrIIA13b blocks the PAM-binding region of Cas9, preventing it from recognizing target DNA.
The 15 amino acid residues at the N terminus of AcrIIA13b are critical for its ability to inhibit Cas9.
Acr proteins serve as counteractive mechanisms against CRISPR-Cas systems in bacteria and archaea.
Understanding AcrIIA13b's structure may help clarify the regulatory mechanisms of CRISPR-Cas systems.

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The CRISPR-Cas systems of adaptive immunity in bacteria and archaea provide resistance against phages and other mobile genetic elements. Counteractive anti-CRISPR (Acr) proteins in phages and archaeal viruses impede these CRISPR-Cas systems. Although CRISPR-Cas systems have revolutionized genome editing, potential off-target events remain a safety concern. Hence, a thorough comprehension of the structural and molecular basis of diverse Acrs is imperative to unravel the fundamental mechanisms governing CRISPR-Cas regulation. Here, we present the structure of AcrIIA13b from Staphylococcus haemolyticus and analyze its structural and functional features to reveal the molecular basis underlying the inhibition of Cas9 by AcrIIA13b. Our structural analysis shows that AcrIIA13b eliminates the cleavage activity of Staphylococcus aureus Cas9 (SauCas9) by blocking the PAM-binding region of Cas9 so that Cas9 cannot recognize the target DNA. In addition, we demonstrate that the 15 amino acid residues at the N terminus of AcrIIA13b, which were revealed to be important for its dimerization, are critical for its inhibitory activity against Cas9. Our findings shed light on the molecular basis of AcrIIA13b-mediated CRISPR-Cas inhibition and provide valuable insights into the arms race between bacteria and phages.

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Investigating the molecular mechanisms underlying the anti‐ CRISPR function of AcrIIA13b protein

Abstract

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