Alternative functions of the CRISPR-associated endonuclease Cas9

我们最近对病原体脑膜炎奈瑟菌II型CRISPR通路的表征揭示了一种流线型的功能性CRISPR- cas结构和一种新的与加工无关的crRNA生物发生模式。II型系统仅利用介导双链DNA断裂（DSBs）的单一效应蛋白Cas9，已发展成为体外rna引导DNA切割和体内基因组编辑的系统。我们将利用奈瑟菌Cas9机制来调节脑膜炎奈瑟菌的基因表达。为了实现基因组沉默（CRISPRi），保留dna结合活性的Cas9核酸酶突变体将被重新编程以抑制或终止转录。为此，将应用与启动子序列或基因编码序列互补的crRNAs。在该项目的第二部分，我们计划研究cas9介导的转录后控制。最近在土拉弗朗西斯菌中的一项研究表明，Cas9与tracrRNA和一个小的CRISPR-Cas相关RNA （scaRNA）一起抑制脂蛋白mRNA。为了分析这种Cas9介导的基因调控是否在其他生物中保守，我们将寻求鉴定neisserial Cas9蛋白的新RNA靶点。为此，我们将结合内源性flag标记Cas9和RNA-seq的交联免疫沉淀（CLIP）实验。此外，ms2标记的tracrRNA和crRNAs将用于鉴定RNA和蛋白质的相互作用伙伴。每个单一CRISPR组件（Cas9， crrna和tracrRNA）的奈瑟菌突变体将被生成，并使用我们完善的RNA-seq方法分析潜在的基因失调。

Our recent characterization of the type II CRISPR pathway in the pathogen Neisseria meningitidis has revealed a streamlined functional CRISPR-Cas architecture and a novel processing-independent mode of crRNA biogenesis. The type II system utilizes only a single effector-protein Cas9, which mediates double-stranded DNA breaks (DSBs), and has been developed into a system for RNA-guided DNA cleavage in vitro and genome editing in vivo. We will utilize the neisserial Cas9 machinery to modulate gene expression in Neisseria meningitidis. To achieve genome silencing (CRISPRi), Cas9 nuclease mutants that retain DNA-binding activity will be re-programmed to repress or terminate transcription. To this end, crRNAs complementary to either a promoter-sequence or the coding sequence of a gene will be applied. In a second part of this project, we plan to investigate Cas9-mediated post-transcriptional control. A recent study in Francisella tularensis showed that Cas9 together with tracrRNA and a small CRISPR-Cas associated RNA (scaRNA) represses a lipoprotein mRNA. To analyze whether such Cas9-mediated gene regulation is conserved in other organisms, we will seek to identify new RNA targets of the neisserial Cas9 protein. To this end, we will combine cross-linking Immunoprecipitation (CLIP) experiments of endogenous FLAG-tagged Cas9 and RNA-seq. In addition, MS2-tagged tracrRNA and crRNAs will be used to identify RNA and protein interaction partners. Neisseria mutants of each single CRISPR component (Cas9, crRNAs and tracrRNA) will be generated and analysed for potential misregulations of genes by using our well established RNA-seq approach.