Dissecting the target recognition by CRISPR-Cas complexes using single-molecule nanomechanical measurements
Prof. Ralf Seidel, Universität Leipzig
Datum: 31.01.2020 um 15:30 Uhr
Ort: Kleiner Physik-Hörsaal N 020, Fakultät für Physik, LMU
The recently discovered CRISPR-Cas enzymes are promising tools in biotechnology and medicine. These enzymes can be guided to any genetic locus by a short RNA that recognizes desired DNA targets by base pairing. The target recognition is, however, highly promiscuous, such that technologically undesired mispairs between RNA and target DNA occur. We employ single-molecule nanomechanical measurements [1] and single-molecule fluorescence imaging [2] to resolve the dynamics of the target recognition process with few base pair resolution [3,4]. We find that this process occurs in sequential base pair steps. Mispairs act as barriers that stall the recognition but that can be overcome by thermal fluctuations. We show that the target recognition process can be quantitatively described by a Markov process in a simplified energy landscape, which can even be accessed experimentally. We think that our quantitative understanding of the targeting process can help to avoid the recognition of wrong targets by CRISPR-Cas complexes in emerging applications.
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