Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus.
Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus.
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作者：
Peng W;Li H;Hallstrøm S;Peng N;Liang YX;She Q
细菌和大肠杆菌编码成簇的、规则间隔的短回文重复序列（CRISPR）系统，以赋予对侵入性病毒和质粒的适应性免疫。最近对CRISPR系统的研究表明，不同的CRISPR相关（Cas）干扰模块通常共存于不同的生物体中，但cas基因的功能尚未在任何这些系统中被剖析。Crenarchaeon Sulfolobus islandicus编码三种不同的CRISPR干扰模块，包括IA型系统和两种IIIB型系统：Cmr-α和Cmr-β。为了研究该生物体中原型间隔区邻近基序（PAM）依赖性DNA靶向活性和成熟CRISPR RNA（crRNA）产生的遗传决定因素，构建了缺失IA型系统的单个基因或去除每个其他Cas模块的突变体。对这些突变体的表征表明，Cas 7、Cas 5、Cas 6、Cas 3 ′和Cas 3”对于PAM依赖的DNA靶向活性是必需的，而Csa 5沿着所有其他Cas模块对于在泉古菌中的靶向活性是必需的。Cas 6被认为是用于前crRNA加工的唯一酶，并且crRNA成熟不依赖于DNA靶向活性。重要的是，我们表明Cas 7和Cas 5分别对于稳定加工中间体和成熟crRNA是必不可少的，并且耗尽Cas 3的解旋酶或核酸酶结构域导致加工中间体的积累。这表明除了Cas 6之外，古细菌IA型系统的其他Cas蛋白也有助于crRNA加工。
Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-α and Cmr-β. To study the genetic determinants of protospacer-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3′ and Cas3” are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show that Cas7 and Cas5 are essential for stabilizing the processing intermediates and mature crRNAs, respectively, and that depleting the helicase or nuclease domain of Cas3 leads to the accumulation of processing intermediates. This demonstrates that in addition to Cas6, other Cas proteins of an archaeal type IA system also contribute to crRNA processing.