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Publications (4 of 4) Show all publications
Camsund, D., Lawson, M. J., Larsson, J., Jones, D., Zikrin, S., Fange, D. & Elf, J. (2020). Time-resolved imaging-based CRISPRi screening. Nature Methods, 17(1), 86-92
Open this publication in new window or tab >>Time-resolved imaging-based CRISPRi screening
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2020 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 17, no 1, p. 86-92Article in journal (Refereed) Published
Abstract [en]

DuMPLING (dynamic mu-fluidic microscopy phenotyping of a library before in situ genotyping) enables screening of dynamic phenotypes in strain libraries and was used here to study genes that coordinate replication and cell division in Escherichia coli. Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2020
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-406174 (URN)10.1038/s41592-019-0629-y (DOI)000508582900040 ()31740817 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2017.0291Knut and Alice Wallenberg Foundation, 2016.0077EU, European Research Council, 616047Swedish Research Council, 642-2013-7841Swedish Research Council, 2016-06213
Available from: 2020-03-06 Created: 2020-03-06 Last updated: 2020-03-06Bibliographically approved
Lawson, M. J., Camsund, D., Larsson, J., Baltekin, Ö., Fange, D. & Elf, J. (2017). In situ genotyping of a pooled strain library after characterizing complex phenotypes. Molecular Systems Biology, 13(10), Article ID 947.
Open this publication in new window or tab >>In situ genotyping of a pooled strain library after characterizing complex phenotypes
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2017 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 13, no 10, article id 947Article in journal (Refereed) Published
Abstract [en]

In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E.coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

Keywords
DuMPLING, live cell, microfluidic, single cell, strain libraries
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-342924 (URN)10.15252/msb.20177951 (DOI)000416160000004 ()29042431 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilEU, European Research Council
Available from: 2018-02-26 Created: 2018-02-26 Last updated: 2018-02-26Bibliographically approved
Jones, D., Leroy, P., Unoson, C., Fange, D., Curic, V., Lawson, M. J. & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science, 357(6358), 1420-1423
Open this publication in new window or tab >>Kinetics of dCas9 target search in Escherichia coli
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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
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-337092 (URN)10.1126/science.aah7084 (DOI)000411880800052 ()28963258 (PubMedID)
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-01-25Bibliographically approved
Lawson, M. J., Petzold, L. & Hellander, A. (2015). Accuracy of the Michaelis–Menten approximation when analysing effects of molecular noise. Journal of the Royal Society Interface, 12(106), 20150054:1-10, Article ID 20150054.
Open this publication in new window or tab >>Accuracy of the Michaelis–Menten approximation when analysing effects of molecular noise
2015 (English)In: Journal of the Royal Society Interface, ISSN 1742-5689, E-ISSN 1742-5662, Vol. 12, no 106, p. 20150054:1-10, article id 20150054Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology Computational Mathematics
Identifiers
urn:nbn:se:uu:diva-248592 (URN)10.1098/rsif.2015.0054 (DOI)000353359900018 ()
Projects
eSSENCE
Available from: 2015-04-01 Created: 2015-04-01 Last updated: 2017-12-04Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-2868-733x

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