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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
116
36-POS
Board 18
Kinetics of dCas9 Target Search in
Escherichia Coli
Daniel Jones
, Cecilia Unoson, Prune Leroy, Vladimir Curic, Johan Elf.
Uppsala University, Uppsala, Sweden.
How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded
oligonucleotide? To address this question we study the CRISPR-associated protein Cas9 which
can be programmed by guide RNAs to bind essentially any DNA sequence. This targeting
flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the
guide RNA. Here we study the search mechanisms of the catalytically inactive dCas9 in
living
Escherichia coli
cells using complementary single molecule fluorescence microscopy and
bulk restriction protection assays. We find that it takes a single dCas9
∼
10 h to find and bind a
specific target, in stark contrast to transcription factors such as LacI, which takes 5 minutes to
locate its target. Thus, the price dCas9 pays for flexibility in targeting is time. By introducing
transcription factor "roadblocks" adjoining dCas9's binding site, we demonstrate that dCas9
undergoes short-range (5-10 bp) sliding along DNA while searching for potential binding sites.
The physical limitations (
i.e.
, slow search) for Cas9 are likely applicable to other systems that are
programmed by single stranded oligonucleotides to locate sequences in dsDNA, such as the
homologous repair machinery.