Print Email Facebook Twitter Visualisation of dCas9 target search in vivo using an open-microscopy framework Title Visualisation of dCas9 target search in vivo using an open-microscopy framework Author Martens, Koen (Wageningen University & Research) van Beljouw, Sam P.B. (Wageningen University & Research) van der Els, Simon (Wageningen University & Research; NIZO food research) Vink, J.N.A. (TU Delft BN/Stan Brouns Lab; Kavli institute of nanoscience Delft) Baas, Sander (Wageningen University & Research) Vogelaar, George A. (Wageningen University & Research) Brouns, S.J.J. (TU Delft BN/Stan Brouns Lab; Kavli institute of nanoscience Delft) van Baarlen, Peter (Wageningen University & Research) Kleerebezem, Michiel (Wageningen University & Research) Hohlbein, Johannes (Wageningen University & Research) Date 2019 Abstract CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox. To reference this document use: http://resolver.tudelft.nl/uuid:a24297fd-3004-4354-b485-3d0986fc7ac2 DOI https://doi.org/10.1038/s41467-019-11514-0 ISSN 2041-1723 Source Nature Communications, 10 (1) Part of collection Institutional Repository Document type journal article Rights © 2019 Koen Martens, Sam P.B. van Beljouw, Simon van der Els, J.N.A. Vink, Sander Baas, George A. Vogelaar, S.J.J. Brouns, Peter van Baarlen, Michiel Kleerebezem, Johannes Hohlbein Files PDF s41467_019_11514_0.pdf 1.46 MB Close viewer /islandora/object/uuid%3Aa24297fd-3004-4354-b485-3d0986fc7ac2/datastream/OBJ/view