Time-resolved imaging-based CRISPRi screening
Abstract
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.
Additional Information
© 2019 Springer Nature Limited. Received 25 May 2019; Accepted 04 October 2019; Published 18 November 2019. Data availability: The microscopy images and image analysis output associated with the DuMPLING experiments are publicly available at the Image Data Resource (https://idr.openmicroscopy.org/) under the accession number idr0065. Other data from this study are available from the corresponding author upon request. Code availability: The code used to analyze the DuMPLING microscopy images and generate figures associated with the DuMPLING experiments is provided as Supplementary Software. This work was supported by the Knut and Alice Wallenberg Foundation (2017.0291 and 2016.0077), the European Research Council (616047) and the Swedish Research Council (VR)(642-2013-7841 and 2016-06213). We are grateful to I. Barkefors for help with the manuscript and figures, to P. Karempudi for making microfluidic molds and to the Dan Andersson lab for kindly sharing E. coli strains. Author Contributions: J.E. conceived the DuMPLING method and SeqA application. D.C. developed cloning methods, and designed and made the strain library. J.E. and M.J.L. managed the project. D.F. and M.J.L. developed phenotyping methods. J.L. and M.J.L. developed genotyping methods. D.F. built the microscope. J.L. and M.J.L. performed microscopy experiments. S.Z. and D.F. developed the image analysis pipeline. S.Z. and D.F. analyzed the DuMPLING data. D.J. performed repression measurements and NGS growth rate experiments. J.E., M.J.L., D.C. and D.F. wrote the paper with input from all authors. Competing interests: The DuMPLING technology is patented with European patent no. EP3167061 (B1).Attached Files
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Additional details
- Alternative title
- A time-resolved imaging-based CRISPRi screening method
- Eprint ID
- 98278
- Resolver ID
- CaltechAUTHORS:20190828-075031250
- 2017.0291
- Knut and Alice Wallenberg Foundation
- 2016.0077
- Knut and Alice Wallenberg Foundation
- 616047
- European Research Council (ERC)
- 642-2013-7841
- Swedish Research Council
- 2016-06213
- Swedish Research Council
- Created
-
2019-08-28Created from EPrint's datestamp field
- Updated
-
2023-06-01Created from EPrint's last_modified field