Single-molecule analysis of non-canonical Cas9 ribonucleoprotein complexes and characterisation of archaeal solo-Cas4 protein variants

越来越多的证据表明，CRISPR-Cas机制发挥的功能超出了其作为防御系统的典型作用。其中，CRISPR-Cas的组分被认为与内源性基因调控、细菌毒力调控、DNA修复、细胞休眠和感染以及基因组进化有关。我们的目标是利用我实验室建立的基于荧光的单分子方法的力量，帮助理解控制非经典CRISPR-Cas功能的分子原理。在CRISPR-Cas SPP 2141的背景下，我们设想进行以下项目：（i）Cas9变体通常参与crRNA指导的DNA切割。来自Franciscella novicida（Fn）的Cas9可以形成由scaRNA、tracrRNA和blp mRNA组成的不寻常的复合物，其最终导致blp mRNA降解。类似地，来自空肠弯曲杆菌的Cas9变体-迄今为止表征的最小Cas9变体-最近显示也与转录后下调的mRNA相关。在结构和动力学方面，rcanonical crRNA/dsDNA/Cas9复合物分别与scaRNA/tracrRNA/mRNA/FnCas 9和crRNA/tracrRNA/mRNA/CjCAs 9复合物有何不同？单分子FRET测量将被用来回答这个紧迫的问题。(ii)Cas 1来自E.大肠杆菌和激烈毕赤酵母已显示与参与DNA修复的蛋白质（例如RecB、RecC、RvuB和UvrC）相互作用。使用单分子下拉（SimPull）和smFRET的组合，我们可以直接从大肠杆菌中分离这些复合物，而不需要体外重建，能够确定这些复合物的化学计量，并可以使用smFRET确定作用机制。最先进的超分辨率显微镜和粒子跟踪实验，我们将确定Cas 1在体内的定位和相互作用。

There is growing evidence that CRISPR-Cas machineries fulfil functions beyond their canonical role as defence systems. Among others, components of CRISPR-Cas are thought to be implicated in endogenous gene regulation, regulation of bacterial virulence, DNA repair, cell dormancy and infection and genome evolution. We aim to contribute to the understanding of the molecular principals that govern non-canonical CRISPR-Cas functions exploiting the power of fluorescence-based single-molecule methods that are established in my laboratory. In context of the CRISPR-Cas SPP2141, we envisage to carry out the following projects: (i) Cas9 variants are usually involved in crRNA-directed DNA cleavage. Cas9 from Franciscella novicida (Fn) can form an unusual complex composed of scaRNA, tracrRNA and the blp mRNA which eventually leads to blp mRNA degradation. Similarly, the Cas9 variant from Campylobacter jejuni – the smallest Cas9 variant characterised so far – was recently shown to be also associated with mRNAs that are posttrancriptionally downregulated. How do the rcanonical crRNA/dsDNA/Cas9 complexes differ from the scaRNA/tracrRNA/mRNA/FnCas9 and crRNA/tracrRNA/mRNA/CjCAs9 complexes, respectively, in terms of structure and dynamics? Single-molecule FRET measurements will be employed to answer this burning question. (ii) Cas1 from E. coli and P. furiosus have been shown to interact with proteins involved in DNA repair (e.g. RecB, RecC, RvuB and UvrC). Using a combination of the single-molecule pulldown (SimPull) and smFRET, we can directly isolate these complexes from E.coli without the need for in vitro reconstitution, are able to determine the stoichiometry of these complexes and can determine the mechanism of action using smFRET. State-of-the-art super-resolution microscopy and particle tracking experiments we will determine the localisation and interactions of Cas1 in vivo.