Elucidation of the molecular mechanism of Cas-endonucleases from bacteria and cyanobacteria

阐明细菌和蓝细菌 Cas 核酸内切酶的分子机制

• 批准号: 405856574
• 负责人: Dr. Sabine Schneider
• 金额: --
• 依托单位: Department Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405856574?language=en
• 关键词: Elucidation; molecular; mechanism; Cas; endonucleases

CRISPR-Cas systems (CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats, Cas= CRISPR-associated gene) are used by prokaryotes to defend themselves against phages, invading nucleic acids and mobile genetic elements. These adaptive immune systems consist of a genomic CRISPR-locus, which is transcribed in the crRNA (CRISPR-RNA) and one (or a complex) effector nuclease, that cleaves the target nucleic acid in a crRNA-sequence dependent manner. The RNA-guided, sequence-dependent cleavage of double stranded DNA by the Cas9 endonuclease of Streptococcus pyogenes (SpyCas9) is currently exploited for the site-specific genome modification of various organisms and has revolutionised the field of biology. Structural biology played a crucial role to engineer and harness the SpyCas9-system as a universal genome editing tool by deciphering its molecular mechanism. About 100 structures of Cas-proteins and CRISPR-associated proteins have been determined by now. However, prokaryotes and archaea possess numerous, highly diverse Cas-systems, with to date unknown natural functions and mechanisms of action as well as a large number of such systems still remaining to be discovered. Based on bioinformatics analysis of bacterial and archaeal genomes, many novel subtypes of Cas-proteins were predicted and classified in bacteria, archaea and cyanobacteria. Furthermore, recent studies show that an active Class 1 CRISPR-Cas system generates oligoadenylate secondary messengers, resulting in the activation of another endonuclease and RNA-degradation. In addition, current data suggest that CRISPR-Cas systems are not merely prokaryotic defence mechanisms, but also play a role in DNA repair, regulation of gene expression, virulence and horizontal gene transfer. This clearly emphasis that despite the tremendous progress in the characterisation of these intriguing systems which has already been made, a lot of information on their molecular mechanisms and functions in the natural organisms is largely missing. My research group focuses on the structure-function relationship of nucleic acids and their recognition by protein factors and small molecules. In this proposed project we want to biochemically and structurally characterise Class 2 Cas-proteins from cyanobacteria and bacteria. This will lead to a detailed elucidation of the molecular mechanisms of these bacterial and cyanobacterial Cas-proteins. In order to obtain a full picture of the atomic details and their function in the natural host, we will cooperate with the groups of G. Bange, W. Hess, G. Klug and A. Marchfelder within the framework of this priority program as outlined in the present proposal. Within the funding period novel Cas- and Cas-associated proteins with particular roles in their natural organisms might be discovered by members of the priority program. Here we will be able to bring our expertise into play and to decipher the molecular mechanisms of these proteins.

原核生物利用 CRISPR-Cas 系统（CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats，Cas = CRISPR 相关基因）来防御噬菌体、入侵核酸和移动遗传元件。这些适应性免疫系统由在 crRNA (CRISPR-RNA) 中转录的基因组 CRISPR 基因座和一种（或复合）效应核酸酶组成，该效应核酸酶以 crRNA 序列依赖性方式切割靶核酸。化脓性链球菌的 Cas9 核酸内切酶 (SpyCas9) 对双链 DNA 进行 RNA 引导、序列依赖性切割，目前已用于多种生物体的位点特异性基因组修饰，并彻底改变了生物学领域。结构生物学通过破译 SpyCas9 系统的分子机制，在设计和利用 SpyCas9 系统作为通用基因组编辑工具方面发挥了至关重要的作用。目前已确定约100种Cas蛋白和CRISPR相关蛋白的结构。然而，原核生物和古细菌拥有众多、高度多样化的Cas系统，其自然功能和作用机制至今未知，还有大量此类系统仍有待发现。基于细菌和古细菌基因组的生物信息学分析，在细菌、古细菌和蓝细菌中预测并分类了许多新的Cas蛋白亚型。此外，最近的研究表明，活跃的 1 类 CRISPR-Cas 系统会产生寡腺苷酸第二信使，从而导致另一种核酸内切酶的激活和 RNA 降解。此外，目前的数据表明，CRISPR-Cas系统不仅仅是原核防御机制，还在DNA修复、基因表达调节、毒力和水平基因转移中发挥作用。这清楚地强调，尽管在这些有趣的系统的表征方面已经取得了巨大进展，但关于它们在自然生物体中的分子机制和功能的大量信息在很大程度上缺失。 我的研究小组专注于核酸的结构功能关系以及蛋白质因子和小分子的识别。在这个拟议的项目中，我们希望对蓝细菌和细菌的 2 类 Cas 蛋白进行生化和结构表征。这将详细阐明这些细菌和蓝细菌 Cas 蛋白的分子机制。为了全面了解原子细节及其在自然宿主中的功能，我们将在本提案概述的优先计划框架内与 G. Bange、W. Hess、G. Klug 和 A. Marchfelder 小组合作。在资助期内，优先计划的成员可能会发现在自然生物体中具有特殊作用的新型 Cas 和 Cas 相关蛋白。在这里，我们将能够发挥我们的专业知识并破译这些蛋白质的分子机制。