Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24

Abstract

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.

AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain

“…Indeed, while numerous Acrs are known to inhibit the activity of Cas9 as monomers, it has been demonstrated previously that certain Acrs exert their function in a dimeric configuration ( 12 , 45 ). In the case of AcrIIA28, the SEC result showed that AcrIIA28 was eluted between the molecular sizes of 17 and 1.35 kDa by producing one prominent peak in the chromatogram (Figure 1A ).…”

“…Upon recognizing a complementary invader DNA sequence, the complex formed by the crRNA and Cas proteins catalyzes the cleavage of the invader's genetic material autonomously or by enlisting supplementary Cas proteins ( 7–10 ). Given the precision with which this bacterial immune system can incise specific DNA sites, CRISPR–Cas systems have found application in gene editing, enabling targeted modifications of genes for both experimental and medicinal objectives ( 11 , 12 ).…”

Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems

“…In genomes of P. aeruginosa phages, regulatory mini-arrays were found within a known anti-CRISPR locus that encodes AcrIF23 and AcrIF24 (68). AcrIF24 binds the Cascade complex, preventing the recruitment of Cas3 (77-79), whereas AcrIF23 inhibits the nuclease activity of Cas3 (80). Combining these Acrs with distinct CRISPR inhibition mechanisms with a regulatory guide that down-regulates Cascade expression is consistent with a multipronged anti-CRISPR strategy that involves both inhibition of the activity of Cas proteins and prevention of further cas gene expression as recently demonstrated for phages evading type V systems (81).…”

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding

“…In contrast to the typical arrangement where acr and aca genes are separate within the same operon, certain Acrs are fusion proteins that combine both canonical Acr and Aca components: one domain responsible for inhibiting the CRISPR-Cas system, and the other domain containing an HTH motif. Several such bi-functional Acrs have been identified, including AcrIIA1 9 , 24 , 25 , AcrIIA6 16 , 26 , AcrIIA13-15 27 , and AcrIF24 28 – 30 .…”