Prokaryotic CRISPR–Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids¹. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR–Cas subtype². Furthermore, although acr genes are frequently organized together in phage-associated gene clusters³, how such inhibitors initially evolve has remained unclear. Here we investigated the Acr content and inhibition specificity of diverse Listeria isolates, which naturally harbour four CRISPR–Cas systems (types I-B, II-A, II-C and VI-A). We observed widespread antagonism of CRISPR, which we traced to 11 previously unknown and 4 known acr gene families encoded by endogenous mobile elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins, one of which assembles into a defective interference complex. Surprisingly, an additional type I-B Cas homologue did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system by means of CRISPR RNA (crRNA) degradation. By probing viral sequence databases, we detected abundant orphan cas genes located within putative anti-defence gene clusters. Among them, we verified the activity of a particularly broad-spectrum cas3 homologue that inhibits type I-B, II-A and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution by cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.

原核 CRISPR-Cas 免疫被抗 CRISPR （Acrs） 破坏，当在噬菌体裂解周期中表达或从常驻原噬菌体或质粒表达时，抗 CRISPR 会抑制 Cas 蛋白活性¹。Acrs 通常与特定的同源 Cas 蛋白结合，因此抑制通常仅限于单个 CRISPR-Cas 亚型²。此外，尽管 acr 基因经常在噬菌体相关基因簇中组织在一起³，但这些抑制剂最初是如何进化的尚不清楚。在这里，我们研究了不同李斯特菌分离株的 Acr 含量和抑制特异性，这些分离株天然含有四种 CRISPR-Cas 系统 （I-B、II-A、II-C 和 VI-A 型）。我们观察到 CRISPR 的广泛拮抗作用，我们将其追溯到由内源性移动元件编码的 11 个以前未知的 acr 基因家族和 4 个已知的 acr 基因家族。其中包括两种与 I-B 型 Cas 蛋白具有序列同源性的 Acr，其中一种组装成有缺陷的干扰复合物。令人惊讶的是，额外的 I-B 型 Cas 同源物并不影响 I 型免疫，而是通过 CRISPR RNA （crRNA） 降解抑制了靶向 RNA 的 VI 型 CRISPR 系统。通过探测病毒序列数据库，我们检测到了位于推定的抗防御基因簇内的大量孤儿 cas 基因。其中，我们验证了一种特别广谱的 cas3 同系物的活性，该同源物抑制 I-B 、 II-A 和 VI-A CRISPR 免疫。我们的观察结果提供了通过 cas 基因共选择进化 Acr 的直接证据，以及具有广谱控制基因组编辑技术潜力的新基因。