Regensburg 2019 – scientific programme
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BP: Fachverband Biologische Physik
BP 12: Poster II
BP 12.68: Poster
Tuesday, April 2, 2019, 14:00–16:00, Poster B2
Correlated Single Molecule Twist and Fluorescence Measurements on CRISPR-Cas Systems — •Pierre Aldag1, Julene Madariaga2, Inga Songailiene3, Virginijus Siksnys3, and Ralf Seidel1 — 1Peter Debye Institute for Soft Matter Physics, University of Leipzig — 2Centro Nacional de Biotecnología (CSIC), Madrid — 3Institute of Biotechnology, Vilnius University
CRISPR-Cas systems are RNA-guided ribonucleoprotein (RNP) complexes with nuclease activity that provide prokaryotes with an adaptive defense mechanism against foreign nucleic acids. The RNP complex recognizes complementary target sites by base-pairing its RNA component with one of the strands of the target DNA while displacing the other one forming a so-called R-loop structure. Considering the vast potential of CRISPR-Cas systems in gene editing technology, it is crucial to fully understand the mechanism behind the targeting process by these enzymes. Here, we employed a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule force and fluorescence spectroscopy measurements. The magnetic tweezers allow us to probe the R-loop formation of the CRISPR system. Using fluorescently-labelled Cascade complexes we are able to additionally follow association and dissociation events prior to the actual R-loop formation. These measurements reveal information about the timescales of the target search as well as about the efficiencies of the search under varying torque conditions. This leads to a better understanding of the target recognition mechanisms by CRISR-Cas enzymes.