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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
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Biology
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JoVE Journal Biology
Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

DOI:
10.3791/52118-v

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09:40 min

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January 03, 2015

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, ,
1Harvard Medical School, 2Division of Hematology/Oncology,Boston Children’s Hospital, 3Department of Pediatric Oncology,Dana-Farber Cancer Institute, 4Howard Hughes Medical Institute

Chapters

  • 00:05Title
  • 01:22Transfecting CRISPRs into MEL Cells
  • 02:35Fluorescence Activated Cell Sorting of Transfected Cells
  • 03:41Primer Validation and Screening Bulk Cultures for CRISPR/Cas9-Mediated Deletion
  • 05:26Screening CRISPR/Cas9 Clones for Deletions and Clone Selection
  • 07:10Results: Deletion of Pim1 in MEL Cells
  • 09:00Conclusion

Summary

Automatic Translation

Automatic Translation

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Tags

Genomic DeletionsCRISPR/Cas9Genome EngineeringSingle Guide RNA (sgRNA)Double-strand Breaks (DSBs)Non-homologous End Joining (NHEJ)Loss-of-function StudiesMammalian Cell Lines

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