Seminar “Single-molecule investigation and modeling of target recognition by CRISPR-Cas complexes”
Date
Thursday, 28th September 2017
Time
12:00 am
Place
University of Barcelona
Faculty of Physics Building
Room 3.20, 3rd floor
Speaker
Dr Ralf Seidel, Leipzig University (Germany)
Abstract
The recently discovered CRISPR-Cas enzymes are promising tools in biotechnology and medicine. Central part of these enzyme systems are large protein complexes harboring a short RNA. The RNA promotes recognition of complementary nucleic acid targets by base pairing. The target recognition is, however, highly promiscuous, such that multiple mispairs between RNA and target strand are allowed. This causes technologically undesired off-target binding.
Here we investigate the recognition of off-target sites by the CRISPR-Cas surveillance complex Cascade using single-DNA twisting experiments. This allows us to resolve the dynamics of the RNA base-pairing with the target. We find that the target recognition is a dynamic zipping process between RNA and DNA. Upon reaching a mismatch site, the zipping stalls and the zipper intermediate frequently collapses.
Eventually thermal fluctuations allow to overcome single mismatches. Upon full zipping of the RNA along the target, a conformational change within the effector complex is triggered that causes DNA degradation. Thus, the complex acts simply as a guard that verifies whether the zipping was successful. Based on these observations we developed a random walk model that can quantitatively describe the dynamics of the target recognition process.
