Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species

Abstract

CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

“…Hence a genome-editing technique reliant on the native CRISPR/Cas machinery for one strain might not work in other closely related strains. Recent studies across a wide-range of bacteria have revealed that anti-CRISPR proteins to silence the native CRISPR/Cas system are also often encoded on the chromosome (37). Although no anti-CRISPR proteins have been detected in Methanosarcina, it is possible that they exist and might potentially complicate use of the native CRISPR/Cas machinery for genome editing.…”

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

“…There are many possible explanations, and we do not claim to have an authoritative answer. However, virus-encoded anti-CRISPRs antagonize CRISPR-mediated immune systems and are expected to drive immune system diversification (41,57,58). Bondy-Denomy et al showed that the anti-CRISPR protein AcrF3 binds to Cas2/3 and blocks recruitment to the Csy complex, and recently determined structures of AcrF3 bound to Cas2/3 explain how the AcrF3 proteins blocks DNA access to the RecA domains of the Cas3 helicase.…”

“…DNA degradation by Cas2/3 is essential for crRNA-guided protection from viral infection, and viruses have evolved suppressors that inhibit the CRISPR immune response (39)(40)(41)(42). One of these suppressors, anti-CRISPR protein 3 (AcrF3), binds to Cas2/3 (18,19,(39)(40)(41)(42)(43).…”

“…One of these suppressors, anti-CRISPR protein 3 (AcrF3), binds to Cas2/3 (18,19,(39)(40)(41)(42)(43). Biochemical data and recent structural models indicate that AcrF3 blocks Cas2/3 recruitment to the target-bound Csy complex, but the impact of this interaction on Cas2/3 cleavage of R-loops has not been tested.…”

New CRISPR-Cas systems discovered

“…What is the missing component(s) that accounts for the inactivity of these archaeal Cas9 proteins? In this regard, recent studies have reported several inhibitors for Class 1 Type I-F (Csy Cascade) [10,11], and Class 2 Type II (Cas9) [12,13] and Type V-B (Cas13b) systems, as well as an activator for Type V-B system (Cas13b) [14], suggesting that there might also be certain regulator(s) or cofactor(s) required for cleavage in archaeal CRISPR-Cas systems. Alternatively, auto-inhibitory processes may control the cleavage activity of these archaeal Cas9 proteins.…”