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Kinetics of dCas9 target search in Escherichia coli

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Science  29 Sep 2017:
Vol. 357, Issue 6358, pp. 1420-1424
DOI: 10.1126/science.aah7084

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Flexible association comes at a price

The CRISPR-Cas9 genome editing system is guided to its target DNA sequence by a small RNA that must search the genome to find its target site. To allow the guide RNA to bind, Cas9 must unwind the DNA at each location that it searches. Jones et al. used single-molecule fluorescence methods and bulk biochemistry to show that Cas9 takes 6 hours to find its target sequence, with each potential target bound for less than 30 ms. Overall, the CRISPR-Cas9 system pays for its flexible association mechanism with slow kinetics, but this can be overcome by using high concentrations of Cas9 and guide RNA.

Science, this issue p. 1420

Abstract

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA 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 Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

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