Figure 2. CRISPR-Cas9-mediated AhR gene editing boosts HIV-1 replication in memory CD4⁺ T cells.

The experimental flow chart in Figure 1A was used to investigate the effect of CRISPR-Cas9-mediated AhR gene editing on HIV-1 replication. Briefly, TCR-activated memory CD4⁺ T cells were electroporated with AhR crRNA or negative crRNA in the presence of the tracrRNA. 3 days after electroporation, cells were exposed to HIV_THRO for 3 h, washed, and cultured for 9 days in the presence of rhIL-2 (5 ng/mL).

(A–C) Shown are cell-associated integrated HIV-DNA levels at day 3 post-infection (n = 4, donors 1–4) (A) and HIV-p24 levels in cell-culture supernatants at days 3, 6, and 9 post-infection in one representative donor (B) and statistical analysis of HIV-1 replication at day 9 post-infection in donors 1–8 (C). Finally, cells were harvested at day 9 post-infection and stained on the surface with CD4 Abs and intracellularly with HIV-p24 Abs.

(D and E) Shown are the effects of AhR gene editing on the frequency of CD4^lowHIV-p24⁺ T cells in each individual donor (D) and statistical analysis in donors 5–8 (E).

(F) The viability dye Aqua Vivid was used to determine cell viability (Vivid−) at day 9 post-infection in donors 5–8.

Paired t test p values are indicated in (A), (C), (E), and (F). Shown are bars that indicate mean values (A), (C), (E), and (F).