Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

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.

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