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

Methanosarcina mazei菌株Gö1编码两个CRISPR-Cas系统，分为亚型I-B和亚型III-C。基于我们在第一个资助期的发现，我们假设Cas6b-IB核糖核酸内酶活性对两种亚型（I-B和III-C）的crRNA成熟至关重要，并且在正常生长条件下，马泽氏分枝杆菌的CRISPR系统通常受到抑制，这可能是由于asrna介导的转录和/或转录后调控。在第二个资助期，我们将重点关注以下主要目标：(i)阐明两种亚型Cas6b核糖核酸内切酶在体内的交叉互补功能，以及通过生化和遗传方法研究天然复合物中Cas6-IB的潜在相互作用伙伴。我们将通过Northern blot和RNA-Seq分析分别表征每种Cas6b蛋白的单缺失突变株（delta-cas6b-IB; delta-cas6b-IIIC），研究它们对体内两个CRISPR位点的crRNA成熟的影响。此外，在诱导启动子控制下表达完整的I-B型cas操纵子亚型的菌株以及缺失选定cas基因的衍生物中，将分析Cas6b-IB蛋白复合物在体内形成和crRNA成熟的可能性。（ii）通过遗传学和生物化学方法揭示CRISPR相关DNA结合蛋白（MM565）以及已鉴定的asRNAs asCas6和asCas3的功能，以阐明它们对马氏分枝杆菌CRISPR活性的调节功能。（iii）为了确定M. mazei - b亚型的PAM序列，并深入了解M. mazei CRISPR系统的进化，我们将在几个新的M. mazei分离株（大约30株）中筛选CRISPR位点上的新间隔物，并利用生物信息学工具研究间隔物的获取。我们还将特别筛选来自我们最近发现的马氏分枝杆菌特异性病毒MSV的间隔物。在用该病毒挑战选定的马氏分枝杆菌菌株后，将进一步追踪从MSV基因组获得的潜在间隔序列。（iv）利用鉴定出的PAM序列，我们的目标是建立基于质粒的合成CRISPR阵列，进一步对I-B亚型进行功能分析，并阐明先导序列的功能。此外，我们建议建立一个研究原间隔物对马泽尔氏菌质粒稳定性影响的系统和一个用于马泽尔氏菌基因沉默的CRISPRi工具。

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.