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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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JoVE Journal Genetics
Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

DOI:
10.3791/59780-v

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00:09 min

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September 04, 2019

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, , , ,
1Donnelly Centre,University of Toronto, 2Department of Molecular Genetics,University of Toronto, 3Institute for Biomaterials and Biomedical Engineering,University of Toronto

Chapters

  • 00:04Title
  • 01:36Pooled CRISPR sgRNA Lentiviral Library Plasmid Amplification
  • 02:24Large-scale CRISPR sgRNA Library Lentivirus Production
  • 03:47Primary Screen Infection, Selection, and Cell Passaging
  • 05:41CRISPR Sample Preparation and Sequencing
  • 06:43Results: Genome-scale CRISPR Screening
  • 07:54Conclusion

Summary

Automatic Translation

Automatic Translation

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Tags

Keywords: CRISPRGenetic ScreensMammalian CellsDrop-out ScreensGuide LibrariesEssential GenesCancerDrug MechanismsCell LinesLentivirusAntibiotic SelectionPlasmid TransformationElectrocompetent CellsLBCarbenicillinPlasmid Purification293T CellsTransfection

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