CRISPR-Cas functions beyond defence in haloarchaea

自从十多年前作为原核免疫系统被发现以来，越来越多的数据表明，CRISPR-Cas系统或其组分除了防御功能外还具有额外的细胞作用。这些额外的功能包括调节毒力，群体行为，胁迫耐受性和基因表达等过程，以及在DNA修复中的作用和对生态学和进化的影响。在上一个资助期，我们研究了CRISPR-Cas在嗜盐古菌Haloferax volcanii中防御之外的功能。我们可以证明CRISPR-Cas参与调节内源基因表达的小RNA s479的成熟和功能。此外，我们收集的数据表明Cas 1参与氧化应激修复。我们还开始分析最近鉴定的CRISPR-Cas变体，HRAMP（盐古菌重复相关神秘蛋白）系统。此外，我们研究了自靶向诱导的基因组切割，揭示了DNA片段是从整个基因组中产生的，并作为新的间隔区并入CRISPR阵列中。在下一个资助期，我们希望进一步研究CRISPR-Cas在内源性基因调控中的潜在作用。Cascade蛋白的共纯化实验将显示mRNA和crRNA样sRNA是否结合，并揭示Cascade与crRNA和sRNA一起是否在内源基因调控中具有活性。我们将详细研究s479与Cascade及其靶mRNA相互作用的分子机制，并继续研究CRISPR-Cas系统与细胞修复途径的相互作用。为此，我们将更详细地研究Cas 1在DNA修复中的功能，并确定直接相互作用伙伴。此外，我们还将研究自我靶向诱导的裂解是如何修复的，以及CRISPR-Cas组分是否参与了修复反应。DNA修复和重组酶在干扰和适应反应中的参与也将被分析。作为第三个目标，我们将研究非经典的CRISPR-Cas变体HRAMP。我们想确定HRAMP蛋白是否形成复合物，以及这种复合物是否结合DNA或RNA。在这个项目的框架内，我们希望确定盐古菌CRISPR-Cas系统的新功能。(1)我们希望确定Cas 1、Cas 2和Cas 4蛋白的其他功能。(2)我们将研究孤儿CRISPR-cas基因。几种盐古菌只含有CRISPR基因或不完整的cas基因组。我们将分析这些基因是否仍然活跃，以及它们具有什么样的功能。(3)我们希望通过CRISPR-Cas系统阐明内源基因的调控。CRISPR基因座可以含有与自身基因组匹配的间隔区。我们想确定这些间隔区是否可以调节内源基因的表达。(4)据报道，细菌中的生物膜形成受CRISPR-Cas调控。我们将研究CRISPR-Cas在盐古菌中对生物膜形成的调控。

Since its discovery as prokaryotic immune system more than ten years ago, more and more data show that the CRISPR-Cas system or its components have additional cellular roles beyond the defence function. These additional functions encompass regulation of processes like virulence, group behaviour, stress tolerance and gene expression as well as roles in DNA repair and impacts on ecology and evolution. In the previous funding period we investigated CRISPR-Cas functions beyond defence in the halophilic archaeon Haloferax volcanii. We could show that CRISPR-Cas is involved in the maturation and function of the small RNA s479 that regulates expression of an endogenous gene. Furthermore, we collected data that suggest involvement of Cas1 in oxidative stress repair. We also initiated the analysis of a recently identified CRISPR-Cas variant, the HRAMP (haloarchaeal repeat associated mysterious protein) system. In addition, we investigated self-targeting induced genome cleavage revealing that DNA fragments are generated from the entire genome and are incorporated as new spacers into the CRISPR arrays.In the next funding period we want to further investigate the potential role of CRISPR-Cas in endogenous gene regulation. Copurification experiments with Cascade proteins will show whether mRNAs and crRNA-like sRNAs are bound and reveal whether Cascade together with crRNAs and sRNAs is active in endogenous gene regulation. We will study the molecular mechanism of the interaction of s479 with Cascade and its target mRNA in detail.We also want to continue our studies on the interaction between the CRISPR-Cas system and the cellular repair pathways. To this end we will investigate the function of Cas1 in DNA repair in more detail and identify direct interaction partners. In addition, we will examine how self-targeting induced cleavages are repaired and if CRISPR-Cas components are involved in the repair reactions. The involvement of DNA repair and recombination enzymes in interference and adaptation reactions will also be analysed.As a third goal we will investigate the non-canonical CRISPR-Cas variant HRAMP. We want to determine whether the HRAMP proteins form a complex and whether this complex binds DNA or RNA. Furthermore we want to identify the targets of this system.In the frame of this project we want to identify novel functions of haloarchaeal CRISPR-Cas systems. (1) We want to determine additional functions of the Cas1, Cas2 and Cas4 proteins. (2) We will investigate orphan CRISPR-cas genes. Several haloarchaeal organism contain only CRISPR genes or an incomplete cas gene set. We will analyse whether these genes are still active and what kind of function they have. (3) We want to unravel the regulation of endogenous genes by the CRISPR-Cas system. CRISPR loci can contain spacers matching the own genome. We want to determine whether these spacers can regulate the expression of the endogenous genes. (4) Biofilm formation in bacteria has been reported to be regulated by CRISPR-Cas. We will investigate the regulation of biofilm formation by CRISPR-Cas in haloarchaea.