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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
JoVE Journal
Genetics
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JoVE Journal Genetics
Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

DOI:
10.3791/57278-v

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08:27 min

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April 10, 2018

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, , , ,
1Stem Cells & Regenerative Medicine Center,Baylor College of Medicine, 2Center for Cell and Gene Therapy,Baylor College of Medicine, 3Centro di Ricerca Emato-Oncologica (CREO),University of Perugia, 4Department of Molecular & Human Genetics,Baylor College of Medicine, 5Texas Children’s Hospital & Houston Methodist Hospital

Chapters

  • 00:05Title
  • 01:05sgRNA Fwd Primer Design, DNA Template Synthesis, and ln Vitro Transcription of sgRNA
  • 03:38Cas9-sgRNA Complexing and Electroporation
  • 05:45Results: Use of Cas9-sgRNA Electroporation Protocol to Study Efficient Knockout of Target Gene
  • 07:48Conclusion

Summary

Automatic Translation

Automatic Translation

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Tags

Keywords: CRISPR/Cas9Gene DisruptionHematopoietic Progenitor CellsRibonucleoproteinSingle-guide RNAPCRT7 RNA PolymeraseGene KnockoutPrimary CellsHigh EfficiencyFast TurnaroundCost-effectiveNormal And Malignant HematopoiesisPrimary T-cellsHematopoietic Cell LinesPrimary Leukemia Samples

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