Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor

Abstract

The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric αβββαα fold comprising three antiparallel β strands flanked by three α-helices and a short 310-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the β1–β2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the β1–β2 and α2–α3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 μM, which compares to KD ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.

Cell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

“…Upon co-delivery of vectors encoding Cas9, sgRNA and Acr, the anti-CRISPR proteins will be made in parallel to Cas9 and the sgRNA. Due to the high affinity of Acrs to Cas9–sgRNA complexes (79,80), this switch design is tighter than the previous system (27). In fact, we showed that by increasing Acr vector doses, Cas9 activity in the OFF state can be pushed towards the detection limits of the used assays (Figures 2A–D, 3 and 4).…”

Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity

“…Anti-CRISPR (Acr) proteins, i.e., naturally occurring CRISPR-Cas inhibitors (20,21), offer flexible strategies to regulate CRISPR-Cas activity and thereby to enhance the specificity of genome perturbations. Among these is AcrIIA4, a potent inhibitor of the Streptococcus pyogenes (Spy)Cas9 (22), which binds Cas9-sgRNA complexes with subnanomolar affinity (23) and impairs DNA targeting as well as nuclease function (24). Recent data show that administering AcrIIA4 shortly after SpyCas9-sgRNA delivery can reduce OFF-target editing (25), likely due to the slower editing kinetics at OFF-target sites as compared to perfect targets (26,27).…”

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system

“…Interestingly, the pre-expressed AcrIIA4 completely inhibited cleavage by SpCas9 even when diluted by a factor of 100. Given that AcrIIA4 is known to act stoichiometrically by directly binding SpCas9 [15,55], these findings would suggest that the levels of preexpressed AcrIIA4 are at least 100-fold higher than those of either SpCas9 or the sgRNA.…”