Figure 5
From: Negative Feedback Regulation of HIV-1 by Gene Editing Strategy
Expression of LTR-Cas9/gRNA protects cells from new HIV-1 infection.
(a,b) Jurkat cells were co-transduced with LV-gRNA A/B and Lenti-LTR(−80/+66)-Cas9-Blast. The next day, cells were infected with HIV-1NL4–3-GFP-P2A-Nef at MOI 0.01. At days 3 and 5 of infection cells were harvested and the level of excision was assessed by LTR specific PCR using genomic DNA as a template (Panel a) and quantified (Panel b) as in Fig. 3. (c) Sequencing analysis of the 205 bp DNA fragment after cloning in TA vector and selection of 10 clones designated at truncLTR 1–10, illustrating the positions of excision fragments compared to the control NL4–3. The positions of gRNAs corresponding to LTR A and LTR B as well as PAM sequences and the primers used for amplification of the DNA are highlighted. (d) Agarose gels depicting results from PCR analysis for the DNA segment corresponding to 5′-UTR, Env and control β-actin DNA in the experimental cells after 3 and 5 days of HIV-1 infection. (e) Results from flow cytometry quantifying the percentage of GFP-positive cells (indicative of viral expression) at three different times post infection. Quantitative detection of viral DNA (f) and viral RNA (g) corresponding to the Gag sequence by Taqman in which β-globin (for DNA) and β-actin (for RNA) were used as a reference. The asterisks (*) illustrate dimerized primers.
