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

Mol Cell. 2019 Feb 7;73(3):611-620.e3. doi: 10.1016/j.molcel.2018.11.011. Epub 2018 Dec 31.

Abstract

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.

Keywords: AcrIIA2; SpyCas9; anti-CRISPR protein; sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophages / genetics
  • Bacteriophages / metabolism*
  • Binding Sites
  • Binding, Competitive
  • CRISPR-Associated Protein 9 / antagonists & inhibitors
  • CRISPR-Associated Protein 9 / chemistry
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / virology
  • Gene Editing / methods*
  • Models, Molecular
  • Molecular Mimicry*
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Structure-Activity Relationship
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Viral Proteins
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes