A targeted CRISPR screen identifies ETS1 as a regulator of HIV-1 latency
Fig 3
CRISPR validation of targets in HIV-1 infected primary CD4 T cells.
(A) Schematic overview of experimental design for CRISPR-Cas9 targeting of host genes in HIV-GFP infected primary CD4 T cells. Activated CD4 T cells were infected with HIV-GFP [26]. At 3 days post infection, infected (GFP+) cells were enriched by magnetic sorting and cultured for 4 days before nucleofection of Cas9/gRNA complexes targeting AAVS1, SMC3, CDK6, ZIC5, FOXE3, SAMD12, ZNF740, ETS1, and TNFAIP3 or a non-targeting gRNA (NT). Pools of 3 different gRNAs were used for each target. HIV-GFP infected cells are denoted in green color. Differential intensity of green color represents latent and active infection. (B) Relative level of HIV-1 expression measured as GFP using flow cytometry. GFP expression was measured at two weeks post nucleofection. Data are displayed as GFP expression in fold change normalized to cells nucleofected with NT gRNA. Each condition represents three biological replicates/donors (Yellow, Blue and Purple) and three technical replicates for each donor. (C) Dot plots representing viral GFP expression at two weeks post knockout. (D) Western blot of the host cellular factors targeting CDK6, ZIC5, ZNF740, ETS1, and TNFAIP3 or a control non-targeting (NT) Cas9/gRNA nucleofected primary cells at one week post nucleofection (pKO). Statistical analysis was conducted for cumulative data. Error bars represent standard deviations, and P values displayed were determined by two-way ANOVA Tukey’s multiple comparisons test. WT, wildtype; NT, non-target; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9.