Inhibition of CRISPR-Cas systems by mobile genetic elements

Abstract

Clustered, regularly interspaced, short, palindromic repeats (CRISPR) loci, together with their CRISPR-associated (Cas) proteins, provide bacteria and archaea with adaptive immunity against invasion by bacteriophages, plasmids, and other mobile genetic elements. These host defenses impart selective pressure on phages and mobile elements to evolve countermeasures against CRISPR immunity. As a consequence of this pressure, phages and mobile elements have evolved 'anti-CRISPR' proteins that function as direct inhibitors of diverse CRISPR-Cas effector complexes. Some of these CRISPR-Cas complexes can be deployed as genome engineering platforms, and anti-CRISPRs could therefore be useful in exerting spatial, temporal, or conditional control over genome editing and related applications. Here we describe the discovery of anti-CRISPRs, the range of CRISPR-Cas systems that they inhibit, their mechanisms of action, and their potential utility in biotechnology.

Figure 1

CRISPR interference machineries that are known to be subject to anti-CRISPR inhibition. CRISPR repeats and spacers are shown as black diamonds and white boxes, respectively, and cas genes are given as colored chevrons. (a) Type I CRISPR-Cas systems. A representative CRISPR-cas locus (subtype I-F) from Pseudomonas aeruginosa strain PA14 is depicted; five other subtypes (I-A through I-E, not shown) have also been defined with variations in cas gene content. Several Cas proteins, along with processed crRNA, assemble into the Csy complex, which can recognize its crRNA-complementary, PAM-flanked DNA target. R-loop formation enables association of the Cas3 effector (dark blue; in this case, fused to Cas2), which cleaves the non-complementary DNA strand. ATP-driven translocation, combined with additional DNA cleavage events, lead to target destruction. (b) Type II CRISPR-Cas systems. A representative CRISPR-Cas locus (subtype II-A) from Streptococcus pyogenes strain SF370 is depicted; two other subtypes (II-B and II-C, not shown) have also been defined with variations in cas gene content and crRNA biogenesis events. Cas9 (yellow), along with processed crRNA and tracrRNA, form a complex that can recognize the crRNA-complementary, PAM-flanked DNA target. Cas9 then cleaves both DNA strands, and this double-strand break leads to target DNA destruction.

Figure 2

Anti-CRISPRs can act at distinct stages of Type I and Type II interference pathways. (a) Three Type I anti-CRISPRs (AcrF1, AcrF2, and AcrF3 have been characterized mechanistically in vitro. AcrF1 and AcrF2 associate with the Csy complex and prevent its association with target DNA; in contrast, AcrF3 (as a homodimer) binds to the Cas2-3 effector, preventing its association with the DNA-bound Csy complex. (b) One Type II-C anti-CRISPR (AcrIIC3_Nme) and two Type II-A anti-CRISPRs (AcrIIA2_Lmo and AcrIIA4_Lmo) have each been shown to prevent the functional output of dCas9’s target DNA association within cells, indicating that they prevent DNA binding.

Figure 3

Established strategies for identifying novel categories of anti-CRISPRs. (a) Many validated acr genes are tightly linked with aca genes (thought to encode transcriptional regulators of acr expression). Searches for aca orthologs can reveal novel, flanking acr genes, especially in genomic regions associated with phage-like or MGE-like sequences. Some of these novel acr genes appear in the genomes of bacteria that harbor CRISPR-Cas systems of different types (e.g., Type II). (b) In many cases, PAM-flanked protospacers are not observed in the same chromosome as matched CRISPR spacers, because CRISPR targeting of the host chromosome can lead to cell death. Expression of an anti-CRISPR (e.g. by an integrated prophage or MGE), however, would be expected to prevent self-targeting-induced cell death. Therefore, bacterial genomes with self-targeting spacers suggests the presence of anti-CRISPR expression. The acr gene that prevents autoimmune cell death can then be identified by association with prophage-like or MGE-like sequences, tight aca linkage, and other genomic/bioinformatic criteria, as well as by functional tests of CRISPR interference inhibition.