RNA processing and activation of Type IIIA CRISPR-Cas systems

DNA靶向I型和II型CRISPR-Cas系统通过与其crrna的互补碱基配对来识别其核酸靶标。crRNA-DNA靶配对始于原间隔邻近基序（PAM），进一步的杂交以类似拉链的方式进行。一旦到达目标的PAM远端，构象变化（I型系统锁定，II型系统参与hnh结构域）触发裂解因子招募或裂解本身。两者，PAM识别连同PAM-远端验证因此允许在其全长上对目标进行验证。III型系统的效应复合物与其crRNA结合到单链RNA靶标上，并迅速降解结合的RNA。此外，crRNA和靶标之间的充分匹配会触发Cas10亚基的HD结构域（一种非特异性单链dna酶）的激活。III型效应复合物被认为针对来自入侵DNA的新转录本，并同时降解转录本和DNA。然而，与I型和II型系统相比，人们对如何选择降解目标知之甚少。鉴于I型和III型系统之间的结构相似性，我们假设这两种系统在目标识别机制上具有相似性。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，并了解该复合体如何在RNA切割后长时间保持激活状态。为此，我们将采用共聚焦和宽视场单分子荧光实验以及单分子力学测量。

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.