Multiple nucleic acid cleavage modes in divergent type III CRISPR systems.
Multiple nucleic acid cleavage modes in divergent type III CRISPR systems.
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DOI：
10.1093/nar/gkw020
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发表时间：
2016-02-29
影响因子：
14.9
通讯作者：
White MF
中科院分区：
文献类型：
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作者：
Zhang J;Graham S;Tello A;Liu H;White MF
CRISPR-Cas是一种RNA引导的适应性免疫系统，可以保护细菌和古菌免受核酸的入侵。III型系统(Cmr，CSM)已被证明在体外切割RNA靶标，其中一些能够转录依赖的DNA靶向。Sulfolobus solfararicus有两个不同的III型系统亚型(SSO-IIID和一个含有Cmr7的SSO-IIIB变种)。在这里，我们报告了SSO-IIID和SSO-IIIB复合体都以尺子机制切割同源RNA靶标，间距为6或12个核苷酸，与Cas7主干的组织有关。因此，这种骨架介导的切割活动在III型系统中似乎是普遍的。SSO-IIIB复合体也被认为具有独特的‘UA’切割模式。体外观察到的主要活性取决于蛋白质和靶RNA的相对摩尔浓度。SSO-IIID复合体在体外可以切割质粒DNA靶标，产生线性DNA产物，其活性依赖于Cas10亚基的环化酶和HD核酸酶结构域，这表明这两个核酸酶活性部位在双链DNA靶标的降解中发挥了作用。
CRISPR-Cas is an RNA-guided adaptive immune system that protects bacteria and archaea from invading nucleic acids. Type III systems (Cmr, Csm) have been shown to cleave RNA targets in vitro and some are capable of transcription-dependent DNA targeting. The crenarchaeon Sulfolobus solfataricus has two divergent subtypes of the type III system (Sso-IIID and a Cmr7-containing variant of Sso-IIIB). Here, we report that both the Sso-IIID and Sso-IIIB complexes cleave cognate RNA targets with a ruler mechanism and 6 or 12 nt spacing that relates to the organization of the Cas7 backbone. This backbone-mediated cleavage activity thus appears universal for the type III systems. The Sso-IIIB complex is also known to possess a distinct ‘UA’ cleavage mode. The predominant activity observed in vitro depends on the relative molar concentration of protein and target RNA. The Sso-IIID complex can cleave plasmid DNA targets in vitro, generating linear DNA products with an activity that is dependent on both the cyclase and HD nuclease domains of the Cas10 subunit, suggesting a role for both nuclease active sites in the degradation of double-stranded DNA targets.