Single-molecule analysis of non-canonical Cas9 ribonucleoprotein complexes and characterisation of archaeal solo-Cas4 protein variants

• Applicant: Professorin Dr. Dina Grohmann
• Subject Area: Biochemistry ; Biophysics ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 405973986

There is growing evidence that CRISPR-Cas machineries fulfil functions beyond their canonical role as defence systems. Among others, components of CRISPR-Cas are thought to be implicated in endogenous gene regulation, regulation of bacterial virulence, DNA repair, cell dormancy and infection and genome evolution. We aim to contribute to the understanding of the molecular principals that govern non-canonical CRISPR-Cas functions exploiting the power of fluorescence-based single-molecule methods that are established in my laboratory. In context of the CRISPR-Cas SPP2141, we envisage to carry out the following projects: (i) Cas9 variants are usually involved in crRNA-directed DNA cleavage. Cas9 from Franciscella novicida (Fn) can form an unusual complex composed of scaRNA, tracrRNA and the blp mRNA which eventually leads to blp mRNA degradation. Similarly, the Cas9 variant from Campylobacter jejuni – the smallest Cas9 variant characterised so far – was recently shown to be also associated with mRNAs that are posttrancriptionally downregulated. How do the rcanonical crRNA/dsDNA/Cas9 complexes differ from the scaRNA/tracrRNA/mRNA/FnCas9 and crRNA/tracrRNA/mRNA/CjCAs9 complexes, respectively, in terms of structure and dynamics? Single-molecule FRET measurements will be employed to answer this burning question. (ii) Cas1 from E. coli and P. furiosus have been shown to interact with proteins involved in DNA repair (e.g. RecB, RecC, RvuB and UvrC). Using a combination of the single-molecule pulldown (SimPull) and smFRET, we can directly isolate these complexes from E.coli without the need for in vitro reconstitution, are able to determine the stoichiometry of these complexes and can determine the mechanism of action using smFRET. State-of-the-art super-resolution microscopy and particle tracking experiments we will determine the localisation and interactions of Cas1 in vivo.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2141: Much more than defence: the multiple functions and facets of CRISPR-Cas