Alternative functions of the CRISPR-associated endonuclease Cas9

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Our recent characterization of the type II CRISPR pathway in the pathogen Neisseria meningitidis has revealed a streamlined functional CRISPR-Cas architecture and a novel processing-independent mode of crRNA biogenesis. The type II system utilizes only a single effector-protein Cas9, which mediates double-stranded DNA breaks (DSBs), and has been developed into a system for RNA-guided DNA cleavage in vitro and genome editing in vivo. We will utilize the neisserial Cas9 machinery to modulate gene expression in Neisseria meningitidis. To achieve genome silencing (CRISPRi), Cas9 nuclease mutants that retain DNA-binding activity will be re-programmed to repress or terminate transcription. To this end, crRNAs complementary to either a promoter-sequence or the coding sequence of a gene will be applied. In a second part of this project, we plan to investigate Cas9-mediated post-transcriptional control. A recent study in Francisella tularensis showed that Cas9 together with tracrRNA and a small CRISPR-Cas associated RNA (scaRNA) represses a lipoprotein mRNA. To analyze whether such Cas9-mediated gene regulation is conserved in other organisms, we will seek to identify new RNA targets of the neisserial Cas9 protein. To this end, we will combine cross-linking Immunoprecipitation (CLIP) experiments of endogenous FLAG-tagged Cas9 and RNA-seq. In addition, MS2-tagged tracrRNA and crRNAs will be used to identify RNA and protein interaction partners. Neisseria mutants of each single CRISPR component (Cas9, crRNAs and tracrRNA) will be generated and analysed for potential misregulations of genes by using our well established RNA-seq approach.