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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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JoVE Journal Genetics
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

DOI:
10.3791/60685-v

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08:20 min

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September 02, 2021

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,
1Cold Spring Harbor Laboratory, 2Faculty of Pharmacy,University of Ljubljana

Chapters

  • 00:04Introduction
  • 01:00Lentiviral Preparation
  • 02:47Determining Virus Titer and Transduction Efficacy with Flow Cytometry
  • 04:27Lentiviral Transduction of Target Cells and Conditional Induction of Cas9 Mediated Gene Editing
  • 06:06Results: Analysis of Reversibility and Rapidity of Destabilized DD-Cas9 Protein
  • 07:24Conclusion

Summary

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Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

Tags

Keywords: CRISPR-Cas9Gene EditingConditional Cas9 StabilizationShield-1Essential GenesTumor Survival GenesDD-Cas9In VivoIn VitroBlood-brain BarrierHEK293T CellsTransfectionVirus SupernatantVirus Titer

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