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

CRISPR-Cas系统(CRISPR=CLUSTED Regulatory Interspace Short Palindrotic Repeats，Cas=CRISPR相关基因)被原核生物用来防御噬菌体、入侵的核酸和可移动的遗传元件。这些适应性免疫系统包括一个基因组CRISPR-基因座，它在crRNA(CRISPR-RNA)中转录，以及一个(或一个复杂的)效应核酸酶，以crRNA序列依赖的方式切割靶核酸。化脓性链球菌Cas9内切酶(SpyCas9)是一种RNA引导的、依赖于序列的双链DNA切割技术，目前已被用于多种生物的定点基因组修饰，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-系统作为一种通用的基因组编辑工具方面发挥了关键作用。到目前为止，已经确定了大约100个Cas蛋白和CRISPR相关蛋白的结构。然而，原核生物和古生物拥有众多的、高度多样化的CAS系统，到目前为止，尚不清楚其自然功能和作用机制，以及大量此类系统仍有待发现。基于细菌和古生菌基因组的生物信息学分析，预测和分类了细菌、古生菌和蓝藻中许多新的Cas蛋白亚型。此外，最近的研究表明，活性的Class1 CRISPR-Cas系统产生寡腺苷二级信使，导致另一种内切酶的激活和RNA的降解。此外，目前的数据表明，CRISPR-Cas系统不仅是原核生物的防御机制，还在DNA修复、基因表达调控、毒力和水平基因转移中发挥作用。这清楚地强调，尽管在表征这些有趣的系统方面已经取得了巨大的进展，但关于它们在自然生物体中的分子机制和功能的许多信息在很大程度上是缺失的。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对它们的识别。在这个拟议的项目中，我们想要从生化和结构上表征蓝藻和细菌中的2类CaS蛋白。这将导致对这些细菌和蓝藻CaS蛋白的分子机制的详细阐明。为了全面了解原子细节及其在自然宿主中的作用，我们将与G.Bange、W.Hess、G.Klug和A.Marchfield小组在本提案中概述的优先计划的框架内进行合作。在资助期内，优先计划的成员可能会发现在其自然生物体中具有特殊作用的新的Cas和Cas相关蛋白质。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。

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.