Crystal structure of the anti‐CRISPR, AcrIIC4

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs)‐CRISPR‐associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti‐CRISPRs (Acrs) that are capable of inhibiting the host CRISPR‐Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti‐CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.

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

“…AcrIIC4 was initially identified in Haemophilus parainfluenzae (Hpa) ( 31 ), and two crystal structures of free AcrIIC4 have been reported. 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.…”

Keeping Cas9 out of the (R-)loop

“…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.…”

“…However, these studies showed ambiguous results in terms of structural conformation and mode of action. Kim et al revealed a monomeric and compact AcrIIC4 conformation in which it forms four short α-helices ( 16 ). Based on the observation that it interferes with DNA binding, it was hypothesized that AcrIIC4 blocks DNA-binding sites in Cas9 ( 16 ).…”

“…Kim et al revealed a monomeric and compact AcrIIC4 conformation in which it forms four short α-helices ( 16 ). 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 ).…”