Prof. Ralf Seidel, Universität Leipzig

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.  

[1] Huhle et al. Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy. Nat. Commun. 6, 5885 (2015)
[2] Kemmerich et al. Simultaneous single-molecule force and fluorescence sampling of DNA nanostructure conformations using magnetic tweezers. Nano Lett. 16, 381-386 (2016)
[3] Rutkauskas et al. Directional R-Loop Formation by the CRISPR-Cas surveillance complex Cascade provides efficient off-target site rejection. Cell Rep. 10, 1534-1543 (2015)
[4] Songaliene et al. Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length. Cell Rep. 28, 3157-3166 (2019)