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.

原核生物使用CRISPR-CAS系统（CRISPR =群集的调节性插入性插入的短质体重复序列，CAS =与CRISPR相关的基因）来防御噬菌体，入侵核酸和移动遗传元素。这些适应性免疫系统由基因组CRISPR-locus组成，该基因组crRNA（CRRNA（CRISPR-RNA）和一个（或复杂）效应子核酸酶在CRRNA序列依赖性方式中切割靶核酸。目前利用Cas9链球菌的Cas9核酸内切酶（SpyCAS9）对RNA引导的序列依赖性DNA裂解（spycas9），用于各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其分子机制来设计和利用SpyCAS9-System作为通用基因组编辑工具而发挥着至关重要的作用。到目前为止，已经确定了大约100种CAS-蛋白质和CRISPR相关蛋白的结构。但是，原核生物和古细菌具有众多高度多样化的cas系统，迄今为止，自然功能和作用机理以及许多此类系统仍在发现。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型CAS-蛋白质的亚型。此外，最近的研究表明，一个活跃的1类CRISPR-CAS系统会产生寡磷酸二苯基二级信使，从而激活另一种核酸酶和RNA降解。此外，当前的数据表明，CRISPR-CAS系统不仅是原核防御机制，而且在DNA修复，基因表达的调节，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取得了巨大进展，但许多关于它们的分子机制和功能的信息在很大程度上是缺失的。 我的研究小组着重于核酸的结构功能关系及其蛋白质因子和小分子的识别。在这个拟议的项目中，我们希望在生化和结构上表征来自蓝细菌和细菌的2类CAS-蛋白质。这将导致对这些细菌和蓝细菌CAS-蛋白质的分子机制的详细阐明。为了完整了解原子细节及其在自然主机中的功能，我们将与G. Bange，W。Hess，G。Klug和A. Marchfelder的群体合作，在本提案中概述的此优先计划的框架内。在资助期内，优先计划的成员可能会发现新颖的CAS和CAS相关蛋白质在其自然有机体中具有特殊作用的蛋白质。在这里，我们将能够使我们的专业知识发挥作用，并破译这些蛋白质的分子机制。