Function and Evolution of archaeal stand-alone cas genes and cas-related anti-CRISPR genes

内切核酸酶Cas 1是CRISPR/Cas系统的两个核心蛋白之一。Cas 1蛋白的一个独特的系统发育组位于新鉴定的称为Casposon的移动的元件上。Casposons是原核生物中第一个自我合成转座子家族。它们显示出靶位点偏好，并且高度类似于Polinton/Maverick超家族的真核DNA转座子。通过对62个不同的马氏甲烷八叠球菌菌株的基因组比较，证明了最近的卡波森移动性的证据。在Mazei甲烷八叠球菌菌株Gö 1的基因组中鉴定的casposon属于编码预测的casposase（casposon编码Cas 1）的家族-2-casposon组。本项目的目的是：（i）阐明新发现的卡泊子的转座机制; 1.我们将描述casposon的特征，特别是关注casposase及其识别和结合先前在M内鉴定的靶位点的能力。利用体外整合酶分析、微量热泳技术以及微型连接子的建立和使用。(ii)将在长期进化实验中使用野生型菌株以及用各种应力挑战的过表达casposase的菌株来研究Casposon迁移率。这将包括产生和使用含有选择标记的修饰的caposon。(iii)几个新的甲烷八叠球菌菌株将被分离和分析的Casposons。(iv)生物信息学分析可用的和新测序的甲烷八叠球菌分离株，沼气反应器的可用宏基因组和相关的元转录组，以及甲烷八叠球菌的群体测序。Mazei与动员casposons将给出一个全面的看法casposons在甲烷八叠球菌的演变。特别是，我们将深入了解不同的casposon单位的进化，到基因组中的casposon插入位点，并诱导基因组重排。总的来说，结合这些实验和生物信息学分析将使新的见解casposons和它们的进化。

The endonuclease Cas1 is one of the two core proteins of CRISPR/Cas systems. A distinct phylogenetic group of Cas1 proteins is located on newly identified mobile elements termed casposons. Casposons represent the presumed first family of self-synthetizing transposons in prokaryotes. They show a target site preference and highly resemble the eukaryotic DNA transposons of the Polinton/Maverick superfamily. Evidence for recent mobility of casposon was demonstrated by genome comparison of 62 different Methanosarcina mazei strains. The casposon identified in the genome of Methanosarcina mazei strain Gö1 belongs to the group of family-2-casposons encoding a predicted casposase (casposon encoded Cas1). In this project we aim to (i) elucidate the transposition mechanism of the newly identified casposon in M. mazei Gö1. We will characterize the casposon, particularly focusing on the casposase and its ability to recognize and bind to the previously identified target sites within the M. mazei genome using in vitro integrase assays, microscale thermophoresis technic and establishing and using a minicasposon. (ii) Casposon mobility will be studied in a long term evolutionary experiment using the wild-type strain as well as a strain overexpressing the casposase challenged with various stresses. This will include the generation and use of a modified caposon containing a selectable marker. (iii) Several new Methanosarcina strains will be isolated and analyzed for casposons. (iv) Bioinformatic analysis of available and newly sequenced Methanosarcina isolates, of available metagenomes and associated metatranscriptomes of a biogas reactor, and of population sequencing of M. mazei with mobilized casposons will give a comprehensive view of the evolution of casposons in Methanosarcina. In particular, we will gain insights into the evolution of the different casposon units, into the casposon insertion sites in the genome, and into the induced genomic rearrangements. Overall combining these experimental and bioinformatic analyses will allow new insights into casposons and their evolution.