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An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9

Alexander P Hynes1, Geneviève M Rousseau1, Marie-Laurence Lemay1

  • 1Département de biochimie, de microbiologie, et de bioinformatique, Faculté des sciences et de génie, Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, QC, G1V 0A6, Canada.

Nature Microbiology
|August 9, 2017
PubMed

Insights

Researchers discovered a novel anti-CRISPR (Acr) protein in a virulent phage that effectively disables CRISPR-Cas immunity in bacteria. This finding expands the known diversity of Acrs and offers new tools for controlling genome editing.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • The CRISPR-Cas system, derived from prokaryotic immunity, is a powerful genome-editing tool.
  • Bacterial viruses (phages) can evade CRISPR-Cas immunity through various mechanisms, including anti-CRISPR (Acr) proteins.
  • Existing Acrs originate from temperate phages or genomic elements and share common properties.

Purpose of the Study:

  • To identify novel anti-CRISPR (Acr) proteins from virulent phages.
  • To characterize the mechanism and scope of activity of a newly identified Acr.
  • To explore the biotechnological potential of Acrs as off-switches for CRISPR-Cas genome editing.

Main Methods:

  • Challenged a Streptococcus thermophilus strain with CRISPR immunity against virulent phages.
  • Identified a phage exhibiting exceptionally high evasion rates (>40,000×).
  • Systematically cloned phage genes to pinpoint the specific Acr responsible for immunity abolition and tested its activity in different strains and bacterial genera using the SpCas9 system.

Main Results:

  • A novel, unrelated Acr was identified in a virulent phage infecting Streptococcus thermophilus.
  • This Acr was solely responsible for abolishing CRISPR-Cas immunity in the tested strain.
  • The Acr demonstrated activity in a different S. thermophilus strain, against unrelated phages, and effectively disabled SpCas9-mediated immunity in another bacterial genus.

Conclusions:

  • A new class of anti-CRISPR (Acr) protein has been discovered in a virulent phage, distinct from previously characterized Acrs.
  • This Acr represents a significant expansion of the known anti-CRISPR repertoire and their evolutionary origins.
  • The identified Acr completely inhibits SpCas9 activity, highlighting its potential as a controllable 'off switch' for CRISPR-based genome editing applications.

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