Genetic Characterization of Antiplasmid Immunity through a Type III-A CRISPR-Cas System
Genetic Characterization of Antiplasmid Immunity through a Type III-A CRISPR-Cas System
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DOI：
10.1128/jb.01130-13
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发表时间：
2014-01-01
影响因子：
3.2
通讯作者：
Marraffini, Luciano A.
中科院分区：
文献类型：
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作者：
Hatoum-Aslan, Asma;Maniv, Inbal;Marraffini, Luciano A.
许多原核生物具有由成簇的规则间隔的短回文重复序列（CRISPR）编码的适应性免疫系统。CRISPR基因座产生小向导RNA（crRNA），其与侧翼CRISPR相关（cas）基因结合，通过反义靶向机制对抗病毒并阻断质粒转移。CRISPR-Cas系统已被分类为三种类型（I至III），其采用不同的crRNA生物发生和靶向机制。表皮葡萄球菌RP 62 a中的III-A型系统阻断葡萄球菌接合质粒的转移，并携带9个cas-csm基因。先前的生物化学分析表明，Cas 10、Csm 2、Csm 3、Csm 4和Csm 5形成含crRNA的核糖核蛋白复合物;然而，这些基因对反质粒靶向的作用仍然未知。在这里，我们确定了抗质粒免疫所需的cas-csm基因，并使用遗传和生化分析来研究这些基因内预测的基序和结构域的功能。我们发现许多突变通过影响Cas Csm复合物或crRNA生物发生的形成来影响免疫力。令人惊讶的是，Cas 10-Csm复合物成员的预测核酸酶结构域中的突变对抗质粒免疫或crRNA生物发生没有可检测的影响。相比之下，csm 6的缺失和cas 10-Csm Palm聚合酶结构域的突变阻止了CRISPR免疫，而不影响复合物的形成或crRNA的产生，这表明它们参与了靶点破坏。通过描述该系统的遗传要求，我们的研究结果进一步有助于对III型CRISPR-Cas系统的机理理解。
Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR- Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA- containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10-Csm Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.