FIG 7.

HEAL knockout maintains HIV-1 suppression after AZT withdrawal. (A) Location of the guide RNA used for CRISPR-Cas9-mediated editing of the HEAL locus in H9 cells. Arrows indicate the primers used to amplify the genomic region harboring the editing site. Red lines indicate the segments amplified and the segments predicted to be present in positive clones after T7EI digestion. (B) Experimental design for the analysis of HIV-1 replication in HEAL^−/− or control cells. H9 T cells were infected with control or HEAL sgRNA-carrying lentiviruses, maintained under puromycin selection conditions for 4 days, and then infected with HIV-1 at an MOI of 0.025 or 0.5. Cells were treated with AZT (20 μM) between days 25 and 28 and then switched to AZT-free or 20 μM AZT-containing medium for an additional 28 days. On day 56, cells were collected and analyzed. (C and D) RT-qPCR analysis of GP120 mRNA in H9 cells infected at an MOI of 0.025 (C) or 0.5 (D). Signals were normalized to GAPDH mRNA levels. n = 3, mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.