Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria
Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria
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DOI：
10.1128/jb.00105-22
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发表时间：
2022-04-07
影响因子：
3.2
通讯作者：
Bondy-Denomy, Joseph
中科院分区：
文献类型：
--
作者：
Govindarajan, Sutharsan;Borges, Adair;Bondy-Denomy, Joseph
簇状规则间隔短回文重复序列(CRISPR)-CRISPR相关(Cas)系统是原核生物适应性免疫系统，已有很好的生化特征，但体内的时空调控和细胞生物学在很大程度上仍未得到解决。在这里，我们使用在染色体CRISPR-Cas基因座引入的荧光融合蛋白来研究I-F型CRISPR-Cas系统在铜绿假单胞菌中的定位。当细胞中缺少靶点时，级联复合体是广泛的核样结合，而Cas3则弥漫在细胞质中。然而，当靶向整合的原噬菌体时，CRISPR RNA(CrRNA)引导的I-F级联复合体和细胞中的大多数Cas3分子被招募到单个焦点。形成级联复合体的csy蛋白的类核结合是crRNA依赖的，并被抗CRISPR AcrIF2的表达特异性地抑制，从而阻断Protspacer相邻基序(PAM)的结合。Cas9核酸酶也是类核的，只有当单引导RNA(SgRNA)结合时才是定位的，这一点可被PAM结合抑制剂AcrIIA4取消。我们的发现揭示了依赖PAM的类核监视和I型CRISPR-Cas的时空调节，它将核酸酶-解旋酶Cas3从crRNA引导的监视复合体中分离出来。CRISPR-Cas效应器在细胞内是如何组织的，目前还不清楚。通过对I-F型CRISPR-Cas系统的细胞生物学研究，我们发现，监视复合体在很大程度上是类核结合的，而Cas3核酸酶是细胞质的。类核定位也是保守的2类CRISPR-CAS单蛋白效应器Cas9。我们对监视复合体和Cas3的差异定位的观察揭示了一种新的翻译后时空调控层，以防止自身免疫。
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor 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.IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.