RNA processing and activation of Type IIIA CRISPR-Cas systems

DNA靶向I型和II型CRISPR-CAS系统通过与它们的crRNAs互补碱基配对来识别它们的核酸靶标。CrRNA-DNA靶标配对在Protspacer相邻基序(PAM)处启动，进一步的杂交以拉链的方式进行。一旦到达靶标的PAM远端，构象变化(I型系统锁定，II型系统参与HNH结构域)就会触发切割因子招募或切割本身。因此，PAM识别和PAM-远端验证一起允许在其全长上进行目标验证。III型系统的效应复合体与其crRNA结合到单链RNA靶标上，并迅速降解结合的RNA。此外，crRNA与靶之间的充分匹配会触发Cas10亚单位的HD结构域的激活，这是一种非特异性的单链DNA酶。III型效应复合体被认为是针对入侵者DNA的新转录物，并同时降解转录物和DNA。然而，与第一类和第二类系统相比，人们对如何选择目标进行降解知之甚少。鉴于I型和III型系统之间的结构相似，我们假设这两个系统在目标识别机制方面具有相似之处。在这里，我们想要解决激活III-A型效应复合体(CSM)的HD结构域的机制，并了解该复合体是如何在RNA切割后长时间保持激活状态的。为此，我们将采用共聚焦和广场单分子荧光实验以及单分子机械测量。

DNA targeting Type I and Type II CRISPR-Cas systems recognize their nucleic acid targets using complementary base pairing with their crRNAs. crRNA-DNA target pairing is initiated at the protospacer adjacent motif (PAM) from which further hybridization proceeds in a zipper-like fashion. Once the PAM distal end of the target is reached, a conformational change (locking by Type I systems, HNH-domain engagement by Type II systems) triggers cleavage factor recruitment or cleavage itself. Both, PAM recognition together with PAM-distal end verification allows hereby the target verification across its full length. Effector complexes of Type III systems bind with their crRNA to single-stranded RNA targets and rapidly degrade the bound RNA. In addition, sufficient matching between crRNA and target triggers the activation of the HD domain, an unspecific single-stranded DNAse, of the Cas10 subunit. Type III effector complexes are thought to target emerging transcripts from invader DNA and to simultaneously degrade both the transcript as well as well as the DNA. Compared to Type I and Type II systems, it is, however, rather poorly understood how targets are selected for degradation. Given the structural similarities between Type I and Type III systems we hypothesize that both systems feature similarities in their target recognition mechanism. Here, we want to resolve the mechanism that activates the HD-domain of the Type III-A effector complex (Csm) and understand how the complex maintains the activated state over long times after RNA cleavage. To this end we will employ confocal and wide-field single-molecule fluorescence experiments as well as single-molecule mechanical measurements.