Visualisation of dCas9 target search in vivo using an open-microscopy framework
Koen Martens (Wageningen University & Research)
Sam P.B. van Beljouw (Wageningen University & Research)
Simon van der Els (NIZO food research, Wageningen University & Research)
Jochem N.A. Vink (TU Delft - BN/Stan Brouns Lab, Kavli institute of nanoscience Delft)
Sander Baas (Wageningen University & Research)
George A. Vogelaar (Wageningen University & Research)
Stan J.J. Brouns (Kavli institute of nanoscience Delft, TU Delft - BN/Stan Brouns Lab)
Peter van Baarlen (Wageningen University & Research)
Michiel Kleerebezem (Wageningen University & Research)
Johannes Hohlbein (Wageningen University & Research)
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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.
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