Choice of Targeted Locus, Guide RNA (gRNA), and Targeting Vector Design
3:08
CRISPR-Cas9-based Targeting of Jurkat Cells
4:36
Generation of Clonal Lines and Screening for Correct Targeting
7:50
Screening of Single-cell Clones by Flow Cytometry and PCR
11:46
Results: Screening and Analysis of Single-cell Clones for Correct Reporter Integration
13:16
Conclusion
Transcript
This method can help answer the question of how HIV provirus integration influences androgynous gene expression, and why certain genomic integration sites are being selected in lately HIV-infected cells. The main advantage of this technique is tha
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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.