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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
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JoVE 杂志 遗传学
Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

DOI:
10.3791/60085-v

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09:04 min

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September 25, 2019

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, , ,
1Center for Cellular and Molecular Therapeutics,The Children’s Hospital of Philadelphia, 2Department of Pathology and Laboratory Medicine,The Children’s Hospital of Philadelphia, 3Department of Pathology and Laboratory Medicine,University of Pennsylvania

Chapters

  • 00:04Title
  • 01:19Test gRNA (GuidE RNA) Cutting Efficiency
  • 03:50Clone Screening
  • 04:51Transfection Setup
  • 06:07Checking for Mutations in Single Colonies
  • 07:11Results: Cutting Efficiency, and Generation and Screening of Mutations
  • 08:10Conclusion

Summary

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This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Tags

CRISPR-Cas9Human Pluripotent Stem CellsIsogenic LinesHeterozygous MutationsGene FunctionDisease ModelingColony PickingFluorescence-activated Cell SortingMEFsROCK Inhibitor

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