Data that underlies all figures.

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here, we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct physical interaction between the 2 proteins. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

噬菌体编码抗CRISPR（Acr）蛋白，可使CRISPR-Cas细菌免疫系统失活，助力噬菌体成功入侵宿主、完成复制并实现原噬菌体整合。Acr蛋白通过多种不同机制抑制CRISPR-Cas系统。AcrIIA1广泛存在于多种噬菌体与质粒中，可特异性结合Cas9的HNH结构域，是首个被发现能够抑制化脓链球菌Cas9（SpyCas9）的Acr蛋白。本研究报道了人类细胞培养体系中AcrIIA1诱导SpyCas9与金黄色葡萄球菌Cas9（SauCas9）发生降解的现象，这是首个在人类细胞中观测到Acr诱导CRISPR-Cas核酸酶降解的案例。若AcrIIA1发生突变，破坏其与SpyCas9之间的直接物理相互作用，则AcrIIA1介导的SpyCas9降解将被完全阻断。AcrIIA1所介导的靶向Cas9蛋白降解，可在人类基因治疗中调控Cas9核酸酶的活性。AcrIIA1分子量小且具有特异性，可被应用于CRISPR-Cas蛋白降解靶向嵌合体（PROTAC）技术，为开发安全精准的基因编辑应用提供了实用工具。