Functional analysis of the CRISPR-Cas systems in Methanosarcina mazei strain Gö1

马氏甲烷八叠球菌 Gö1 菌株 CRISPR-Cas 系统的功能分析

• 批准号: 206969706
• 负责人: Professorin Dr. Ruth Anne Schmitz-Streit
• 金额: --
• 依托单位: Institut für Allgemeine Mikrobiologie
• 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/206969706?language=en
• 关键词: Functional; analysis; CRISPR; Cas; systems

Methanosarcina mazei strain Gö1 encodes two CRISPR-Cas systems, which are classified as subtype I-B and subtype III-C. Based on our findings during the first funding period we hypothesize that endoribonuclease activity of Cas6b-IB is crucial for crRNA maturation of both subtypes (I-B and III-C) and that the CRISPR systems of M. mazei are in general repressed under normal growth conditions potentially due to transcriptional and/or post-transcriptional regulation mediated by asRNAs. In the second funding period we will focus on the following main goals (i) elucidating the in vivo function of the Cas6b endoribonuclease of both subtypes with regard to cross-complementation as well as studying potential interacting partners of Cas6-IB in native complexes by biochemical and genetic approaches. We will characterize the respective single deletion mutant strains of each Cas6b protein (delta-cas6b-IB; delta-cas6b-IIIC) investigating their impact on crRNA maturation of both CRISPR loci in vivo by Northern blot and RNA-Seq analysis. Further, potential in vivo formation of Cas6b-IB protein complexes and crRNA maturation will be analysed in strains expressing the complete cas-operon subtype I-B under the control of an inducible promoter as well as derivatives missing selected cas genes. (ii) Revealing the function of the CRISPR associated DNA binding protein (MM565) as well as of the identified asRNAs, asCas6 and asCas3, by genetic and biochemical approaches in order to clarify their proposed regulatory function regarding the CRISPR activity in M. mazei. (iii) Aiming to identify the PAM sequence(s) for subtype I-B in M. mazei and gain insight in the evolution of the CRISPR systems in M. mazei we will screen for new spacers in the CRISPR loci in several new M. mazei isolates (approximately 30) and investigate for spacer acquisition using bioinformatic tools. We will as well particularly screen for spacers derived from our recently identified M. mazei specific virus MSV. Potential spacer acquisition derived from MSV genome will be further tracked after challenging selected M. mazei strains with the virus. (iv) With the PAM sequences identified we aim to establish a plasmid based synthetic CRISPR array for further functional analysis of subtype I-B and to elucidate the function of the leader sequence. Besides we propose to establish a system to study the effect of protospacers on plasmid stability in M. mazei and a CRISPRi tool for gene silencing in M. mazei.

玛氏甲烷链霉菌Gö1编码两个CRISPR-Cas系统，分为I-B亚型和III-C亚型。根据我们在第一个资助期的发现，我们假设Cas6b-IB的内切核酸酶活性对两个亚型(I-B和III-C)的crRNA成熟至关重要，并且在正常生长条件下，玛泽分枝杆菌的CRISPR系统普遍受到抑制，这可能是由于asRNAs介导的转录和/或转录后调控。在第二个资助期，我们将集中于以下主要目标：(I)阐明两个亚型的Cas6b内切核酸酶在体内的交叉互补功能，以及通过生化和遗传学方法研究Cas6-IB在天然复合体中潜在的相互作用伙伴。我们将通过Northern印迹和RNA-Seq分析来鉴定每个Cas6b蛋白(Delta-Cas6b-IB；Delta-Cas6b-IIIC)各自的单缺失突变株，以研究它们对体内两个CRISPR基因座crRNA成熟的影响。此外，将分析在可诱导启动子控制下表达完整的cas-操纵子亚型I-B的菌株中Cas6b-IB蛋白复合体的体内形成和crRNA成熟的可能性，以及缺失选定的cas基因的衍生物。(Ii)通过遗传和生化方法揭示CRISPR相关DNA结合蛋白(MM565)以及已鉴定的asRNAas Cas6和asCas3的功能，以阐明它们对M.maazei中CRISPR活性的调节功能。(3)为了鉴定玛泽分枝杆菌I-B亚型的PAM序列(S)，并深入了解玛泽分枝杆菌CRISPR系统的进化，我们将在几个新的玛泽分枝杆菌分离株(约30个)的CRISPR基因座中筛选新的间隔区，并利用生物信息学工具研究间隔区的获取。我们还将特别筛选来自我们最近发现的马分枝杆菌特异性病毒MSV的间隔区。在用MSV病毒攻击选定的Mazei菌株后，将进一步追踪来自MSV基因组的潜在间隔区获取。(4)通过对PAM序列的鉴定，我们的目标是建立一个基于质粒的合成CRISPR阵列，用于进一步分析I-B亚型的功能，并阐明前导序列的功能。此外，我们还建议建立一套系统来研究原扩增物对玛泽分枝杆菌中质粒稳定性的影响，并建立一个用于玛泽分枝杆菌基因沉默的CRISPRi工具。