Anti-CRISPR Protein

The anti-CRISPR proteins provide phages with an active counter-defense system that inactivates CRISPR-Cas interference complexes in a sequence-independent manner. CRISPR-Cas RNA-guided immune systems are widespread in prokaryotes, and play a major part in microbial evolution. The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. In the past few years, a variety of phages and other mobile genetic elements have been shown to encode proteins that interact directly with components of the CRISPR-Cas system and inactivate it. Although only a few anti-CRISPR proteins have been studied in detail, it is clear that there are many effective strategies used to inhibit CRISPR-Cas systems.

Anti-CRISPR Protein Families

The first examples of active inhibitors of CRISPR-Cas systems were discovered in a closely related group of Pseudomonas spp. phages. These phages were able to infect and propagate in a Pseudomonas aeruginosa strain with an active type I-F CRISPR system even though they possessed protospacer sequences that should have been targeted by this system. In total, five distinct proteins (AcrF1~AcrF5) were shown to inactivate the type I-F CRISPR system. In a follow-up study, four additional distinct families of small proteins (AcrE1~AcrE4) were shown to inhibit the type I-E CRISPR-Cas system of P. aeruginosa. These proteins were encoded by genes in the same group of phages and positioned adjacent to the type I-F anti-CRISPR genes. Similar to the type I-F anti-CRISPR proteins, no homologues of the type I-E anti-CRISPR proteins were identified in MGEs of other bacterial genera.

Table 1. All Described Anti-CRISPR Protein Families
Anti-CRISPR Protein Family CRISPR System Inhibited Originating Species Number of Amino Acids Known Mechanisms of Activity
AcrE1 Type I-E P. aeruginosa 100
AcrE2 Type I-E P. aeruginosa 84
AcrE3 Type I-E P. aeruginosa 68
AcrE4 Type I-E P. aeruginosa 52
AcrF1 Type I-F P. aeruginosa 78 Inhibits DNA binding
AcrF2 Type I-F P. aeruginosa 90 Inhibits DNA binding
AcrF3 Type I-F P. aeruginosa 139 Prevents Cas3 recruitment by Cascade
AcrF4 Type I-F P. aeruginosa 100
AcrF5 Type I-F P. aeruginosa 79
AcrF6 Type I-E and I-F O. smirnovii 100
AcrF7 Type I-F P. aeruginosa 67
AcrF8 Type I-F Delftia sp. 670 92
AcrF9 Type I-F V. parahaemolyticus 68
AcrF10 Type I-F S. xiamenensis 97 DNA mimic, blocks DNA binding
AcrIIA1 Type II-A L. monocytogenes 149 Blocks DNA binding
AcrIIA2 Type II-A L. monocytogenes 123 Inhibits DNA binding
AcrIIA3 Type II-A L. monocytogenes 125
AcrIIA4 Type II-A L. monocytogenes 87 PAM mimic, inhibits DNA binding
AcrIIA5 Type II-A S. thermophilus 140
AcrIIA6 Type II-A S. thermophilus phage 183
AcrIIC1 Type II-C N. meningitidis 85 Binds HNH domain; prevents cleavage
AcrIIC2 Type II-C N. meningitidis 123
AcrIIC3 Type II-C N. meningitidis 116 Blocks DNA binding; dimerizes Cas9

The nine anti-CRISPR protein families initially identified were restricted to the Pseudomonas genus and did not share any common sequence motifs that could lead to the discovery of new anti-CRISPR proteins. Strikingly, the genomic context of the genes encoding these proteins was very similar. Phages encoding anti-CRISPR proteins also encoded a predicted helix-turn-helix transcriptional regulator, Aca1, that immediately downstream of the anti-CRISPR genes. Aca1 was used as a search query, and the anti-CRISPR activities of proteins encoded next to aca1 orthologs were tested. Five new families of type I-F anti-CRISPR proteins (AcrF6~AcrF10) and three families of type II-C anti-CRISPR proteins (AcrIIC1~AcrIIC3) were discovered subsequently. There are currently 23 unique families of anti-CRISPR proteins that have been identified, but only a few have been studied in detail.

Mechanisms for CRISPR-Cas Inhibition by Anti-CRISPR Proteins

CRISPR-Cas activity has been shown to drive phages to extinction when they are faced with bacterial populations encoding diverse crRNA sequences and mutation alone is their only escape. However, phages encoding anti-CRISPRs were shown to avoid extinction under the same conditions. There are numerous steps of CRISPR-Cas activity that could be subject to inhibition by anti-CRISPR proteins. For example, anti-CRISPRs could prevent the acquisition of new CRISPR spacers, block expression of Cas proteins, inhibit crRNA transcription or processing, prevent the assembly of the active CRISPR-Cas complex, inhibit binding to the foreign DNA element, or block cleavage activity. These strategies of the anti-CRISPR proteins binding to critical catalytic residues and interaction surfaces assure that bacteria cannot escape inhibition and still maintain CRISPR-Cas activity.

Over the past few years, mechanism of action for six anti-CRISPR proteins have been delineated and can be roughly grouped into two mechanistic types: those that disrupt DNA binding and those that inhibit target sequence cleavage. No anti-CRISPRs that affect the expression of Cas genes or expression and processing of crRNAs have been discovered to date. The most common mechanism is interference with DNA binding activity through a direct interaction with the CRISPR-Cas complex. The precise mechanisms by which anti-CRISPRs block DNA binding and allow it to escape recognition vary among the anti-CRISPR proteins. For example, although the type I-F anti-CRISPRs AcrF1 and AcrF2 both prevent DNA binding by Csy complex, they achieve this outcome through different mechanisms.

Mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins

Fig 1. Mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

Studies have shown that AcrF1 and AcrF2 interact with different protein subunits, respectively. Two to three copies of monomeric AcrF1 bind to the Cas7f hexamer, which forms the extended backbone of the type I-F Cascade complex. Interactions are formed between a cluster of three key residues on the surface of AcrF1 and exposed lysine residues in the Cas7f protein backbone, blocking access of the DNA target. By contrast, the small, acidic AcrF2 protein binds to two other proteins (Cas8f-Cas5f) in type I-F Cascade and sterically blocks DNA binding by competing with the target DNA for interactions with two positively charged helices on the neighbouring Cas7f subunits. Similarly, the AcrIIA4 anti-CRISPR protein targets type II-A CRISPR-Cas9 systems by mimicking dsDNA and occupying the PAM-binding site of S. pyogenes Cas9, thereby abrogating the ability of S. pyogenes Cas9 to bind target DNA.

Anti-CRISPR proteins that inhibit target DNA cleavage have been identified for both type I-F and type II-C CRISPR-Cas systems. AcrF3 binds as a dimer to the Cas3 helicase-nuclease protein, covering a very large interface on the Cas3 protein, and thus prevents its recruitment to the DNA-bound CRISPR-Cas complex. This interaction blocks the Cas3 binding sites for both DNA and Cascade and locks Cas3 in an ADP-bound inactive form. Similarly, the type II-C AcrIIC1 protein was shown to bind to the most highly conserved feature of Cas9 proteins, the HNH domain, through critical catalytic residues, inhibiting cleavage of both target and non-target DNA strains. This interaction prevents target strand cleavage by the HNH nuclease domain, maintaining Cas9 in an inactive conformation and blocking the subsequent cleavage of the non-target strand by the RuvC domain.

Applications of Anti–CRISPR Proteins

Owing to the unique mechanisms of action of anti-CRISPR proteins, they can be used in creative ways to modulate CRISPR-Cas function.The potential biotechnological applications of type II anti-CRISPRs are more obvious, given the extensive use of CRISPR-Cas9‑based genome editing technologies. Anti-CRISPRs may provide a valuable ''off switch'' for Cas9 activity for therapeutic uses and gene drives. Studies focused on type II-C and type II-A anti-CRISPR proteins have shown anti-CRISPR proteins that inhibit Cas9 can be harnessed to decrease off-target effects. Anti-CRISPR proteins could also be used to restrict editing activity to particular tissues or developmental stages and to generally increase the safety and efficiency of gene editing technology. Phages used for gene therapy could be outfitted with a variety of anti-CRISPRs to expand their host range and prevent the bacterial adaptive immune response from being mounted. Discovery of anti-CRISPR proteins has opened up a new area of phage research and has provided a valuable addition to the CRISPR toolbox.

Anti-CRISPR Protein Related References

1. Karen L. Maxwell. The Anti-CRISPR Story: A Battle for Survival. Molecular Cell. October 5, 2017; 68:8-14.
2. Bondy-Denomy et al. Multiple mechanisms for CRISPR–Cas inhibition by anti–CRISPR proteins. Nature. 2015 October 1; 526(7571): 136–139.
3. Pawluk et al. Anti-CRISPR: discovery, mechanism and function. NATURE REVIEWS | MICROBIOLOGY. JANUARY 2018; VOLUME 16:12-16.
4. F Bubeck. Engineered anti-CRISPR proteins for optogenetic control of CRISPR–Cas9. NATURE METHODS. October 30, 2018.
5. AP Hynes et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nature Communications. 2018;9:2919.