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CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
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Immunology and Infection
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JoVE Journal Immunology and Infection
CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

DOI:
10.3791/58572-v

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14:27 min

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November 14, 2018

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, , ,
1Heinrich Pette Institute,Leibniz Institute for Experimental Virology, 2Department of Anesthesiology,University Medical Center Hamburg-Eppendorf, 3German Center for Infection Research (DZIF)

Chapters

  • 00:04Title
  • 00:42Choice of Targeted Locus, Guide RNA (gRNA), and Targeting Vector Design
  • 03:08CRISPR-Cas9-based Targeting of Jurkat Cells
  • 04:36Generation of Clonal Lines and Screening for Correct Targeting
  • 07:50Screening of Single-cell Clones by Flow Cytometry and PCR
  • 11:46Results: Screening and Analysis of Single-cell Clones for Correct Reporter Integration
  • 13:16Conclusion

Summary

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We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Tags

Keywords: CRISPR-Cas9Genome EngineeringJurkat Reporter ModelsHIV-1 InfectionProviral Integration SitesGene ExpressionIn-vitro ModelsGuide RNA DesignNCBI BLASTHomology ArmsJurkat T-cell Transfection

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