The CRISPR system at single-molecule level
le 21 avril 2016 à 11 h
Christophe Rouillon, Laboratory of Molecular Biophysics, Université de Leipzig, Allemagne
The recently discovered CRISPR system in prokaryotes is often referred as an adaptive and heritable immune system in the sense that bacteria acquire resistance to viruses by integrating short viral DNA sequences (protospacers) in their genomes as memory of former infections. Further discoveries led to the use of the endonuclease Cas9 from this system to develop molecular tools for genome engineering (editing) in eukaryotic cells. Cas9, effector complex of the Type II CRISPR-Cas system, is a large monomer carrying a microRNA (crRNA) which recognises foreign DNA through base pairing. Fully matched target will then be cleaved by Cas9 dual endonuclease activity, generating a double strand DNA break. In contrast, the interference process of Type I CRISPR-Cas system is mediated by the multimeric ribonucleoprotein surveillance complex Cascade, which then recruits the DNA helicase-nuclease Cas3 for target degradation once the foreign DNA is fully recognised.
In both Type I and Type II CRISPR-Cas systems, DNA R-loop formation is a prerequisite for efficient DNA interference (target DNA degradation). R-loop formation as well as its length and stability can be characterized in real time at single-molecule level using magnetic tweezers, where the amount of DNA untwisting is detected.
Furthermore, we would like to decipher the sequential molecular dynamics of some poorly understood CRISPR mechanisms in Type I systems by coupling our magnetic tweezers with TIRF microscopy. We are also interested in the more sophisticated Type III system that cleaves DNA on top of targeting and cleaving RNA.
Lieu(x) : Amphithéatre Curie, Ecole Polytechnique
Contact : Hannu Myllykallio
hannu.myllykallio at polytechnique.edu