Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

细菌和古细菌的CRISPR-Cas系统可提供针对遗传入侵者（如噬菌体和质粒）的RNA导向免疫。在靶标RNA识别后，III型CRISPR-Cas系统产生环状寡聚腺苷酸第二信使，通过其CARF域激活下游效应子，包括Csm6核糖核酸酶。在这里，我们显示意大利肠球菌Csm6（EiCsm6）降解其关联的环状六-AMP（cA6）活化剂，并报告与cA6模拟物结合的EiCsm6的晶体结构。我们的结构，生化和体内功能检测揭示了CARF结构域对cA6的识别如何激活Csm6 HEPN结构域进行旁链RNA降解，以及CARF结构域介导的cA6裂解如何提供内在的转换，从而限制了Csm6的活性。环核酸酶。这些机制有助于快速清除入侵者并确保终止CRISPR干扰以限制自身毒性。