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

内切酶Cas1是CRISPR/Cas系统的两个核心蛋白之一。Cas1蛋白的一个独特的系统发育群位于新发现的可移动元件上，称为Casposons。Casposon是原核生物中推测的第一类自合成转座子。它们表现出靶点偏好，与Polinton/Maverick超家族的真核DNA转座子高度相似。通过对62株不同的玛氏甲烷链霉菌的基因组比较，证明了酪蛋白最近的迁移性。玛氏甲烷链霉菌Gö1株基因组中鉴定的酪蛋白属于编码预测酪蛋白酶的家族2-酪蛋白(Casposon编码Cas1)。在本项目中，我们的目标是(I)阐明新发现的Mazei Gö1中Casposon的转座机制。我们将利用体外整合酶分析、微型热电泳法和建立和使用微型asposon来表征Casposon的特性，特别是它识别和结合M.mazei基因组中先前确定的目标位置的能力。(Ii)将在长期进化实验中研究Casposon的迁移率，使用野生型菌株以及在各种压力下挑战过表达Casposase的菌株。这将包括产生和使用包含可选择标记的修改后的火锅。(Iii)将分离出几种新的甲烷八叠胞菌，并对其进行酪蛋白分析。(4)对现有的和新测序的甲烷八叠球菌分离株、沼气反应器的可用元基因组和相关的元转录本以及利用动员的酪蛋白对玛氏分支杆菌的种群进行的生物信息学分析将全面了解甲烷八叠球菌中的酪蛋白的进化。特别是，我们将深入了解不同的酪蛋白单位的进化，到基因组中的酪蛋白插入位置，以及诱导的基因组重排。总体而言，将这些实验分析和生物信息学分析结合起来，将使人们能够对酪蛋白及其进化有新的见解。

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.