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Related Experiment Video

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MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
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Optical Pooled Screens in Human Cells.

David Feldman1, Avtar Singh2, Jonathan L Schmid-Burgk2

  • 1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Physics, MIT, Cambridge, MA 02142, USA.

Cell
|October 19, 2019
PubMed
Summary

This study introduces a pooled optical genetic screening method for mammalian cells, enabling image-based phenotyping of complex cellular processes. The novel approach successfully identified genes involved in nuclear factor-kappa B (NF-κB) signaling dynamics.

Keywords:
CRISPRfunctional genomicshigh-content screeningin situ sequencingoptical pooled screenpooled screen

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Area of Science:

  • Cell Biology
  • Genetics
  • Molecular Biology

Background:

  • Genetic screens are essential for identifying genes that control cellular phenotypes.
  • Pooled genetic screens enhance scalability but are incompatible with detailed imaging of dynamic cellular behaviors.
  • Existing methods lack the capacity for high-throughput, image-based screening of pooled genetic libraries.

Purpose of the Study:

  • To develop a pooled optical genetic screening approach for mammalian cells that integrates image-based phenotyping with pooled library demultiplexing.
  • To apply this method to identify genes involved in nuclear factor-kappa B (NF-κB) signaling.
  • To investigate the temporal regulation of NF-κB signaling using live-cell imaging.

Main Methods:

  • Developed a pooled genetic screening method using targeted in situ sequencing for demultiplexing.
  • Screened a library of 952 genes in millions of cells by imaging RelA (p65) nuclear translocation.
  • Utilized live-cell imaging to capture dynamic changes in p65 nuclear retention.

Main Results:

  • Identified 15 known components of the NF-κB pathway in a single-time-point screen across three cell lines.
  • Discovered a role for Mediator complex subunits in regulating the duration of p65 nuclear retention through live-cell imaging.
  • Demonstrated the ability to screen spatially and temporally defined phenotypes using pooled libraries.

Conclusions:

  • The developed pooled optical genetic screening approach enables high-throughput, image-based phenotyping of pooled libraries in mammalian cells.
  • This method is effective for identifying components of signaling pathways and dissecting their temporal dynamics.
  • The approach offers a powerful tool for studying complex cellular phenotypes with unprecedented scale and resolution.