CRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in Type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex.

中文翻译：

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细菌中I型CRISPR复合物和Cas3核酸酶的亚细胞定位

CRISPR-Cas系统是原核生物适应性免疫系统，已在生化方面得到了很好的表征，但体内时空调节和细胞生物学仍未解决。在这里，我们使用荧光融合蛋白研究了铜绿假单胞菌天然的IF型CRISPR-Cas系统的定位。当针对整合的噬菌体时，crRNA引导的（Csy）复合物和细胞中的大部分Cas3分子被募集到一个单一的焦点。然而，当细胞中缺乏靶标时，Csy复合物被广泛地与核苷结合，而Cas3则在细胞质中扩散。Csy蛋白的核样缔合是crRNA依赖性的，并受抗CRISPR AcrIF2的表达抑制，从而阻止PAM结合。仅当gRNA结合时，Cas9核酸酶才被定位在核苷酸上，而PAM模拟物AcrIIA4则将其取消。