The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs

Abstract

Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPR-Cas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action were elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field.

Keywords: CRISPR-Cas; Cas9; anti-CRISPR; bacteriophage.

Figure 1

Characterized and predicted mechanisms for anti-CRISPR protein function. CRISPR-Cas immune function is broken down into five distinct processes, shown in gray boxes. Acr proteins that inhibit these processes are shown for both type I and type II CRISPR-Cas systems. All characterized type I-F Acr proteins (AcrF1–5) have been demonstrated to inhibit both adaptation and immunity by preventing either foreign DNA recognition (AcrF1, AcrF2, and AcrF4) or Cas3 nuclease recruitment (AcrF3). AcrIIA2, AcrIIA4, and AcrIIC3 prevent DNA target binding by Cas9. All anti-CRISPRs are defined by their ability to ultimately prevent foreign DNA destruction, though the mechanisms by which most of them accomplish this task are still unknown. Abbreviations: cr, CRISPR RNA; R, repeat.

Figure 2

Anti-CRISPR (acr) locus organization. Stereotypical organizations of acr loci encoded by phages and mobile genetic elements (MGEs) are shown. Unique acr genes are named and shown in color, whereas non-acr genes are shown in gray and are annotated with predicted functions when possible. (a) Pseudomonas aeruginosa Mu-like phage acr locus. The acr genes are all integrated at the same locus between two highly conserved structural genes (gray) that are homologous to Mu phage gene G and Mu phage protease/scaffold genes (I/Z). Many loci encode both type I-E (AcrE1-4) and I-F (AcrF1-5) Acr proteins, all adjacent to the conserved anti-CRISPR-associated gene 1 (aca1). A representative phage is indicated for each unique locus architecture. Panel adapted from Reference . (b) acr loci in diverse Proteobacteria are shown. These acr loci do not share a common “genomic neighborhood,” but are all anchored by HTH-encoding anti-CRISPR-associated genes (aca1-3). Representatives of each acr-aca association are shown in the indicated species. Panel adapted from Reference . (c) Listeriophage acrIIA locus. The listeriophage locus is near the left end of the integrated prophage genome and a highly conserved endolysin gene (lys). All listeriophage acr loci are anchored by the HTH-encoding gene acrIIA1. Panel adapted from Reference .

Figure 2

Anti-CRISPR (acr) locus organization. Stereotypical organizations of acr loci encoded by phages and mobile genetic elements (MGEs) are shown. Unique acr genes are named and shown in color, whereas non-acr genes are shown in gray and are annotated with predicted functions when possible. (a) Pseudomonas aeruginosa Mu-like phage acr locus. The acr genes are all integrated at the same locus between two highly conserved structural genes (gray) that are homologous to Mu phage gene G and Mu phage protease/scaffold genes (I/Z). Many loci encode both type I-E (AcrE1-4) and I-F (AcrF1-5) Acr proteins, all adjacent to the conserved anti-CRISPR-associated gene 1 (aca1). A representative phage is indicated for each unique locus architecture. Panel adapted from Reference . (b) acr loci in diverse Proteobacteria are shown. These acr loci do not share a common “genomic neighborhood,” but are all anchored by HTH-encoding anti-CRISPR-associated genes (aca1-3). Representatives of each acr-aca association are shown in the indicated species. Panel adapted from Reference . (c) Listeriophage acrIIA locus. The listeriophage locus is near the left end of the integrated prophage genome and a highly conserved endolysin gene (lys). All listeriophage acr loci are anchored by the HTH-encoding gene acrIIA1. Panel adapted from Reference .

Figure 2

Anti-CRISPR (acr) locus organization. Stereotypical organizations of acr loci encoded by phages and mobile genetic elements (MGEs) are shown. Unique acr genes are named and shown in color, whereas non-acr genes are shown in gray and are annotated with predicted functions when possible. (a) Pseudomonas aeruginosa Mu-like phage acr locus. The acr genes are all integrated at the same locus between two highly conserved structural genes (gray) that are homologous to Mu phage gene G and Mu phage protease/scaffold genes (I/Z). Many loci encode both type I-E (AcrE1-4) and I-F (AcrF1-5) Acr proteins, all adjacent to the conserved anti-CRISPR-associated gene 1 (aca1). A representative phage is indicated for each unique locus architecture. Panel adapted from Reference . (b) acr loci in diverse Proteobacteria are shown. These acr loci do not share a common “genomic neighborhood,” but are all anchored by HTH-encoding anti-CRISPR-associated genes (aca1-3). Representatives of each acr-aca association are shown in the indicated species. Panel adapted from Reference . (c) Listeriophage acrIIA locus. The listeriophage locus is near the left end of the integrated prophage genome and a highly conserved endolysin gene (lys). All listeriophage acr loci are anchored by the HTH-encoding gene acrIIA1. Panel adapted from Reference .

Figure 3

Characterized and predicted anti-anti-CRISPR mechanisms. To inhibit CRISPR-Cas immunity, acr genes need to be transcribed and translated inside a host cell during infection; each of these steps could be inhibited by the host. AcrF proteins can lose efficacy when the intracellular concentration of Cas protein targets increases. Cas mutations that lower or abolish Acr binding affinity for the Cas target could also serve to shift the balance in favor of the CRISPR-Cas system, as could protein inhibitors that sequester Acr proteins and prevent them from binding their Cas targets. Lastly, deployment of multiple types of CRISPR-Cas systems is a mechanism by which cells can protect themselves from subtype-specific Acr proteins and may in part explain the accumulation of multiple CRISPR-Cas system types and subtypes in diverse bacteria.