Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins

Alexander P Hynes,Adeline Goulet,Beatrice Amigues,Christophe Fremaux,Yannick Doyon,Philippe Horvath,Dennis A Romero,Daniel Agudelo,Geneviève M Rousseau,Jeremy Loehr,Christian Cambillau,Sylvain Moineau

doi:10.1038/s41467-018-05092-w

Abstract

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.

Highlights

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses can carry anti-CRISPR (Acr) proteins to evade that immunity
Even in the initial discovery of the anti-phage role of CRISPR-Cas systems in bacteria, it was evident that phages had means of bypassing that immunity —a phage with a single point mutation in the 30-nt sequence targeted by the crRNA2, or in the flanking proto-spacer adjacent motif (PAM)[6] could freely infect the CRISPR-immunized bacterial host
Even for the two Acrs detected in virulent phages (AcrIIA4 from L. monocytogenes and AcrIIA5 from S. thermophilus), the majority of their homologs were found in temperate phages[24,25]

Summary

Introduction

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. The Acrs with orthologs in virulent phages are two (AcrIIA4 and AcrIIA5) of the only three (alongside AcrIIA2) to block the activity of the genome-editing tool SpCas[94,26], derived from the type II-A CRISPR-Cas system of Streptococcus pyogenes[22].

Results

Conclusion