Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4

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

“…In one structure, AcrIIC4 is a monomer consisting of a four-helix bundle ( 32 ). By contrast, the other structure showed AcrIIC4 as a homodimer with each monomer consisting of one long and two short α-helices ( 33 ): a strikingly different arrangement from the monomeric structure. Aside from the structural ambiguity, the mechanism of AcrIIC4 also remains unclear.…”

Keeping Cas9 out of the (R-)loop

“…Based on the observation that it interferes with DNA binding, it was hypothesized that AcrIIC4 blocks DNA-binding sites in Cas9 ( 16 ). In contrast, Hwang et al revealed a homodimeric elongated conformation AcrIIC4, in which each AcrIIC4 monomer forms one long and two short α-helices ( 17 ). In contrast to the two other studies, Hwang et al revealed that AcrIIC4 does not strictly inhibit DNA binding.…”

“…To date, six different type II-C-specific inhibitors have been identified (AcrIIC1-AcrIIC6): AcrIIC1 and AcrIIC3 inhibit the nuclease activity of HNH, AcrIIC2 prevents crRNA loading, and AcrIIC5 and AcrIIC6 inhibit DNA binding ( 8 ). The mode of action and structural mechanism by which AcrIIC4 inhibits Cas9 were not completely clear, as some ambiguity was observed in structures and inhibitory effects of AcrIIC4 ( 15 – 17 ).…”

“…In its initial characterization study, it was demonstrated that ActIIC4 inhibits HpaCas9-mediated antiviral defense and that it interferes with DNA binding and cleavage but not crRNA binding ( 15 ). In pursuit of uncovering the mechanism underlying AcrIIC4-mediated Cas9 inhibition, two additional studies focusing on the structure and biochemistry of AcrIIC4 were published ( 16 , 17 ). However, these studies showed ambiguous results in terms of structural conformation and mode of action.…”

“…In contrast to the two other studies, Hwang et al revealed that AcrIIC4 does not strictly inhibit DNA binding. Based on the observation that AcrIIC4 interacts with the REC1 and REC2 domains, it was hypothesized that AcrIIC4 prevents HNH movement, thereby inhibiting target cleavage ( 17 ). Yet, the exact molecular mechanism by which AcrIIC4 inhibits Cas9 remained unclear.…”