Anti-CRISPR protein mediated degradation of Cas9 in human cells

Abstract

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial8 immune systems, allowing successful invasion, replication, and prophage integration. Acr9 proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by10 numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first11 Acr discovered to inhibit SpyCas9. Here we report the observation of AcrIIA1-induced degradation12 of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of13 CRISPR-Cas nucleases in human cells. Optimized expression of AcrIIA1 in human cells provided14 robust inhibition of SpyCas9 editing but had no impact on Type V CRIPSR-Cas12a, consistent15 with its binding site on the HNH domain. Targeted Cas9 protein degradation by AcrIIA1 could16 modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA117 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for18 developing safe and precise gene editing applications.