Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)–Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR–Cas systems protect themselves remains unclear. Here we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally repressed by the CRISPR–Cas effector but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR–Cas system and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR–Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.

原核生物成簇的规则间隔短回文重复序列 （CRISPR）-Cas 系统极易受到噬菌体编码的抗 CRISPR （Acr） 因子的影响。CRISPR-Cas 系统如何保护自身仍不清楚。在这里，我们发现了一种涉及噬菌体衍生的毒性蛋白的广谱抗 CRISPR 策略。这种毒素的转录通常被 CRISPR-Cas 效应子抑制，但当效应子被任何抗 CRISPR 蛋白或 RNA 抑制时，该效应子被激活以停止细胞分裂。我们表明，这种流产的感染样作用有效地从细菌种群中排出 Acr 元件。此外，我们利用这种抗 CRISPR 机制开发了一种针对 CRISPR-Cas 系统的特定 Acr 候选物的筛选方法，并成功鉴定了两种不同的 Acr 蛋白，它们增强了 CRISPR 效应子与非靶 DNA 的结合。我们的数据强调了 CRISPR 抑制毒素在抵消各种类型的 Acr 因子中的广谱作用。我们提出 CRISPR-Cas 的调节功能赋予宿主细胞针对编码 Acr 的遗传入侵者的群体免疫力，无论它们是否靶向 CRISPR。