Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race

Jan 5, 2019Molecular cell

How a Virus Protein Imitates DNA to Compete with the CRISPR Immune System

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Abstract

The crystal structure of the AcrIIA2-SpyCas9-sgRNA complex was determined at 3.3 Å resolution.

AcrIIA2 binds to SpyCas9 at a site similar to where target DNA typically binds.
The interaction occurs at the protospacer adjacent motif (PAM) recognition region of Cas9.
AcrIIA2 prevents double-stranded DNA detection by mimicking DNA and blocking binding.
The binding motif of AcrIIA2 is distinct from that of AcrIIA4.
This study enhances understanding of how AcrIIA2 inhibits SpyCas9's function.

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CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems provide prokaryotic cells with adaptive immunity against invading bacteriophages. Bacteriophages counteract bacterial responses by encoding anti-CRISPR inhibitor proteins (Acr). However, the structural basis for their inhibitory actions remains largely unknown. Here, we report the crystal structure of the AcrIIA2-SpyCas9-sgRNA (single-guide RNA) complex at 3.3 Å resolution. We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. More specifically, AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. Thus, phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4. Our study provides a more detailed mechanistic understanding of AcrIIA2-mediated inhibition of SpyCas9, the most widely used genome-editing tool, opening new avenues for improved regulatory precision during genome editing.

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Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race

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