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

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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.