Project Details
Projekt
Anti-plasmid and CRISPRi activity of a Pseudomonas oleovorans Type IV-A CRISPR-Cas system
Applicant
Professor Dr. Lennart Randau
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Term
from 2018 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 405858350
Archaea and Bacteria have evolved a variety of strategies to cope with foreign nucleic acids. One of these strategies employs CRISPR-Cas systems, which were originally described to mediate adaptive prokaryotic immunity, providing defense against viral attacks. Cas proteins form ribonucleoprotein complexes with CRISPR RNAs (crRNAs). These RNA molecules contain so-called spacer sequences that identify target nucleic acids via base complementarity. Recently, it emerged that CRISPR-Cas systems can repurpose this RNA guidance mechanism as (i) the spacer content of CRISPR elements is highly dynamic and (ii) a large repertoire of divergent Cas proteins was observed. Our group studies two Class 1 CRISPR-Cas ribonucleoprotein complexes with reduced Cas protein content. First, we investigate the Type I-Fv complex of Shewanella putrefaciens, which was shown to target DNA in the absence of commonly observed large and small subunits. The crystal structure of this assembly suggests that unique features of the Cas proteins Cas5fv and Cas7fv compensate for the loss of these subunits. We aim to study the detailed DNA targeting mechanism of this complex and propose to analyze formation of crRNA-DNA interactions using biolayer interferometry and activity assays with Cas protein mutants and target DNA variants. Collaborative efforts aim to investigate the structural and functional involvement of the helicases Cas3 and DinG and the dynamics of DNA scanning. Second, we identified a Type IV CRISPR-Cas system in Aromatoleum aromaticum and produced and purified recombinant Type IV ribonucleoprotein complexes. Type IV CRISPR-Cas does not contain adaptation modules or known DNA nucleases and its cellular function is unknown. We aim to characterize the composition and stoichiometry of the Type IV complexes using gel-filtration and mass-spectrometry approaches. The apparent absence of DNA nucleases suggests that the unknown activity of these CRISPR-Cas systems might not rely on the degradation of target DNA. Therefore, we aim to study the formation and stability of Typ IV complex-mediated crRNA-DNA interactions, so-called R-loops, in vitro and in vivo using footprinting and bisulfite sequencing techniques. The co-occurrence of transposons in the vicinity of Type IV CRISPR-Cas systems indicates that they potentially facilitate crRNA-guided transposition. We aim to test this novel role of CRISPR-Cas systems by following transposon mobility in response to crRNA targeted R-loop formation using next generation sequencing.
DFG Programme
Priority Programmes
