CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity¹. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient^2,3,4,5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation^6,7, but many CRISPR–Cas systems lack Cas4⁸. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited^9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

CRISPR–Cas 适应性免疫系统从入侵的移动遗传元件中捕获 DNA 片段，并将其整合到宿主基因组中，为 RNA 引导的免疫提供模板¹。CRISPR 系统通过区分自身和非自身来维持基因组完整性并避免自身免疫，对于这一过程，CRISPR/Cas1–Cas2 整合酶是必要的，但还不够^2,3,4,5。在一些微生物中，Cas4 核酸内切酶协助 CRISPR 适应^6,7，但许多 CRISPR-Cas 系统缺乏 Cas4 ⁸。在这里，我们展示了 IE 型系统中的一个优雅的替代途径，使用内部 DnaQ 样核酸外切酶 (DEDDh) 来选择和处理 DNA，以便使用原型间隔子相邻基序 (PAM) 进行整合。天然的 Cas1-Cas2/核酸外切酶融合（修剪整合酶）催化协调 DNA 捕获、修剪和整合。CRISPR 修剪整合酶的五个冷冻电子显微镜结构在 DNA 整合之前和整合过程中进行可视化，显示了不对称处理如何生成尺寸限定的、含有 PAM 的底物。在基因组整合之前，PAM 序列由 Cas1 释放并被核酸外切酶切割，将插入的 DNA 标记为自身，并防止 CRISPR 靶向宿主的异常。总之，这些数据支持一个模型，其中缺乏 Cas4 的 CRISPR 系统使用融合或招募^9,10用于忠实获取新 CRISPR 免疫序列的核酸外切酶。