State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
RNA Biol. 2013 May;10(5):738-48. doi: 10.4161/rna.23798. Epub 2013 Feb 7.
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.
细菌和古菌编码成簇、规律间隔、短回文重复（CRISPR）系统，以赋予其对入侵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）干扰模块经常存在于不同的生物体中，但这些系统中尚未剖析 cas 基因的功能。古菌 Sulfolobus islandicus 编码三个不同的 CRISPR 干扰模块，包括一个 IA 系统和两个 IIIB 系统：Cmr-α和 Cmr-β。为了研究该生物体中依赖于原间隔邻近基序（PAM）的 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 系统单个基因或去除其他 Cas 模块的突变体。这些突变体的特征表明 Cas7、Cas5、Cas6、Cas3'和 Cas3"对于 PAM 依赖性 DNA 靶向活性是必需的，而 Csa5 与所有其他 Cas 模块一起对于该古菌的靶向是可有可无的。Cas6 被认为是前 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 Cas7 和 Cas5 分别对于稳定加工中间产物和成熟的 crRNA 是必需的，并且 Cas3 的解旋酶或核酸酶结构域的缺失导致加工中间产物的积累。这表明除了 Cas6 之外，古菌 IA 系统的其他 Cas 蛋白也有助于 crRNA 加工。
