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Kinetics of dCas9 target search in Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6358, p. 1420-1423Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE , 2017. Vol. 357, no 6358, p. 1420-1423
National Category
Biological Sciences
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URN: urn:nbn:se:uu:diva-337092DOI: 10.1126/science.aah7084ISI: 000411880800052PubMedID: 28963258OAI: oai:DiVA.org:uu-337092DiVA, id: diva2:1177689
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg FoundationAvailable from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-01-25Bibliographically approved

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Jones, DanielLeroy, PruneUnoson, CeciliaFange, DavidCuric, VladimirLawson, Michael J.Elf, Johan

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