Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Abstract

SUMMARY Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a counter-measure, numerous phages are known that produce proteins to block the function of Class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used Class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific “anti-CRISPRs” present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

“…In order to reduce unspecific genomic changes by the nickase, CRISPR-Cas9 inhibitory proteins could be expressed to inhibit the activity of Cas9 after genomic editing (35,36). The prophage-encoded inhibitor proteins AcrIIA2 and AcrIIA4, which allow phages to evade the bacterial host's CRISPR/Cas immune system, were found to inhibit Cas9-based targeting in their native host Listeria monocytogenes, as well as Cas9 of Streptococcus pyogenes in bacteria and human cells.…”

Inhibition of CRISPR-Cas systems by mobile genetic elements

“…3b). Rauch et al [41] searched for genome-sequenced, Cas9-containing strains with unusually high frequencies of apparent self-targeting, hypothesizing that these genomes could harbor prophages expressing candidate anti-CRISPRs. This scenario was confirmed to exist in certain strains of Listeria monocytogenes that encode Type II-A CRISPR-Cas systems [41].…”

“…Rauch et al [41] searched for genome-sequenced, Cas9-containing strains with unusually high frequencies of apparent self-targeting, hypothesizing that these genomes could harbor prophages expressing candidate anti-CRISPRs. This scenario was confirmed to exist in certain strains of Listeria monocytogenes that encode Type II-A CRISPR-Cas systems [41]. Subsequent bioinformatics searches and genetic tests identified four prophage-encoded anti-CRISPR genes ( acrIIA1-4 ), each of which sufficed for inhibition of CRISPR interference by L. monocytogenes Cas9 (LmoCas9).…”

“…SpyCas9 and LmoCas9 are 53% identical at the amino acid sequence level, again indicating that this degree of similarity can suffice for cross-species inhibition by a single Acr protein, and reinforcing the likelihood of broad-spectrum Cas9 inhibition by some type II anti-CRISPRs. Finally, both AcrIIA2 Lmo and AcrIIA4 Lmo were shown to inhibit transcriptional repression by promoter-directed dSpyCas9 in E. coli [41], indicating that both Acrs prevent stable, sgRNA-guided DNA binding by dSpyCas9. Three recent reports describe the structure of AcrIIA4 Lmo in complex with sgRNA-loaded SpyCas9, revealing a DNA-mimicking inhibitor that binds to the PAM-interacting region of the SpyCas9/sgRNA complex [44–46], reminiscent of the inhibitory logic exhibited by the type I inhibitor AcrF2 (see above).…”

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.…”