The immunization of bacteria and archaea against invading viruses via CRISPR adaptation is critically reliant on the efficient capture, accurate processing, and integration of CRISPR spacers into the host genome. The adaptation proteins Cas1 and Cas2 are sufficient for successful spacer acquisition in some CRISPR-Cas systems. However, many CRISPR-Cas systems additionally require the Cas4 protein for efficient adaptation. Cas4 has been implied in the selection and processing of spacer precursors, but the detailed mechanistic understanding of how Cas4 contributes to CRISPR adaptation is lacking. Here, we biochemically reconstitute the CRISPR-Cas type I-D adaptation system and show two functionally distinct adaptation complexes: Cas4–Cas1 and Cas1–Cas2. The Cas4–Cas1 complex recognizes and cleaves protospacer adjacent motif (PAM) sequences in 3′ overhangs in a sequence-specific manner, while the Cas1–Cas2 complex defines the cleavage of non-PAM sites via host-factor nucleases. Both sub-complexes are capable of mediating half-site integration, facilitating the integration of processed spacers in the correct interference-proficient orientation. We provide a model in which an asymmetric adaptation complex differentially acts on PAM- and non-PAM-containing overhangs, providing cues for the correct orientation of spacer integration.

中文翻译：

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Cas4–Cas1 是 CRISPR 适应过程中介导半位点间隔子整合的 Protospacer 相邻基序加工因子

通过 CRISPR 适应对细菌和古生菌进行针对入侵病毒的免疫严重依赖于 CRISPR 间隔区的有效捕获、准确处理和整合到宿主基因组中。适应蛋白 Cas1 和 Cas2 足以在某些 CRISPR-Cas 系统中成功获取间隔子。然而，许多 CRISPR-Cas 系统还需要 Cas4 蛋白才能有效适应。Cas4 已隐含在间隔前体的选择和加工中，但缺乏对 Cas4 如何促进 CRISPR 适应的详细机制理解。在这里，我们生化地重构了 CRISPR-Cas 类型 ID 适应系统，并展示了两种功能不同的适应复合物：Cas4–Cas1 和 Cas1–Cas2。Cas4-Cas1 复合物以序列特异性方式识别并切割 3' 突出端中的原型间隔区相邻基序 (PAM) 序列，而 Cas1-Cas2 复合物通过宿主因子核酸酶定义非 PAM 位点的切割。两个子复合体都能够调解半位点整合，促进处理过的间隔物以正确的干扰熟练方向整合。我们提供了一个模型，其中不对称适应复合物不同地作用于含 PAM 和不含 PAM 的悬垂，为间隔整合的正确方向提供线索。促进以正确的干扰熟练方向集成已处理的隔离物。我们提供了一个模型，其中不对称适应复合物不同地作用于含 PAM 和不含 PAM 的悬垂，为间隔整合的正确方向提供线索。促进以正确的干扰熟练方向集成已处理的隔离物。我们提供了一个模型，其中不对称适应复合物不同地作用于含 PAM 和不含 PAM 的悬垂，为间隔整合的正确方向提供线索。