Alternative functions of the CRISPR-associated endonuclease Cas9

CRISPR 相关核酸内切酶 Cas9 的替代功能

• 批准号: 206951410
• 负责人: Professor Dr. Jörg Vogel
• 金额: --
• 依托单位: Institut für Molekulare Infektionsbiologie (IMIB)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206951410?language=en
• 关键词: Alternative; functions; CRISPR; associated; endonuclease

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.

我们最近对脑膜炎奈瑟氏菌的II型CRISPR途径的表征揭示了一个精简的功能CRISPR-Cas结构和一种新的不依赖于加工的crRNA生物发生模式。第二类系统只利用一个单一的效应蛋白Cas9，它介导双链DNA断裂(DSB)，并已发展成为体外RNA引导的DNA切割和体内基因组编辑的系统。我们将利用NeisSerial 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，将应用与基因的启动子序列或编码序列互补的crRNA。在这个项目的第二部分，我们计划研究Cas9介导的转录后调控。最近在土拉方济氏菌中的一项研究表明，Cas9与trrRNA和一个小的CRISPR-Cas相关RNA(SCARNA)一起抑制脂蛋白mRNA的表达。为了分析这种Cas9介导的基因调控在其他生物中是否保守，我们将寻求识别neisSeries Cas9蛋白的新RNA靶标。为此，我们将结合内源标记的Cas9和RNA-seq的交联免疫沉淀(CLIP)实验。此外，MS2标记的trrRNA和crRNAs将用于识别RNA和蛋白质相互作用伙伴。将产生每个单一CRISPR组件(Cas9、crRNAs和trrRNA)的奈瑟氏菌突变体，并使用我们成熟的RNA-SEQ方法分析潜在的基因误调控。