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

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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.