AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation

Abstract

Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.

Keeping Cas9 out of the (R-)loop

“…Similar to the structure by Hwang et al, Sun et al report an elongated homodimeric AcrIIC4 conformation in the absence of HpaCas9. However, it is shown that soluble AcrIIC4 is monomeric and when bound to HpaCas9 monomeric AcrIIC4 assumes the more compact conformation similar to the conformation observed by Kim et al Explaining the ambiguity in the previously reported structural conformations, Sun et al reveal that the long central alpha helix in the elongated structure of AcrIIC4 bends to facilitate AcrIIC4 interactions with HpaCas9 ( 10 ).…”

“…Furthermore, Acrs can be repurposed for controlling Cas protein activity in genome editing or other biotechnological applications ( 9 ). In this issue of PNAS, Sun et al have studied structures of Cas9 bound to Acr protein AcrIIC4 and thereby uncovered a novel mechanism of Cas9 inhibition: preventing complete R-loop formation ( 10 ).…”

“…Sun et al have now resolved structures of free AcrIIC4, the HpaCas9–sgRNA-DNA complex in the absence of AcrIIC4, and two structures of AcrIIC4-bound HpaCas9–sgRNA complexes in the presence and absence of target DNA ( 10 ). Based on their studies, Sun et al not only explain previously made observations but reveal the molecular mechanism of AcrIIC4-based Cas9 inhibition ( Fig.…”

The role of rhizosphere phages in soil health

“…Moreover, phages have evolved anti-CRISPR proteins (Acrs) that inhibit the cleavage of Cas proteins. For example, AcrIIC4 is a broad-spectrum Acr that binds between the two recognition domains of Cas9, REC1 and REC2, restricting the movement of the REC2 domain and thereby maintaining the integrity of its genome (Sun et al 2023b ). Previous studies have demonstrated that phage–bacteria coevolution follows fluctuating selection in the soil microcosms, where phages and bacteria adapt to their contemporary counterparts in time (Gomez and Buckling 2011 ).…”

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

“…Acrs are versatile inhibitors that are typically small and exhibit low homology to other known protein families in both their protein sequences and three-dimensional structures 7 , 8 . Acrs subvert CRISPR-systems through various strategies, including promoting Cas protein degradation 9 , blocking crRNA loading 10 , 11 , preventing DNA recognition 12 – 15 , and obstructing the activation of the Cas protein by preventing the conformational changes of the surveillance complex 16 – 19 .…”