Fig. 4.

Ablation of CCR5 by LentiCRISPR/SaCas9 in primary human CD4⁺ T cells confers HIV-1 resistance. a Disruption of CCR5 in primary CD4⁺ T cells by LentiCRISPR/SaCas9. The primary CD4⁺ T cells were transduced with LentiCRISPR-SaCas9/sgRNA-sgRNA-#6, #8 or control with MOI of 100. After 72 h transduction, cells were harvested for T7E1 assay. b Detection of CCR5 expression in primary CD4⁺ T cells modified by LentiCRISPR/SaCas9-sgRNA-#6. #8 or control. Modified CD4⁺ T cells were lysed and analyzed by western blotting with anti-CCR5 and anti-GAPDH antibodies. c Sanger sequencing of CCR5 gene target loci in modified primary CD4⁺ T cells. d Ablation of CCR5 in CD4⁺ T cells resistance to HIV-1 infection. The CD4⁺ T cells edited by SaCas9/sgRNA-#6, #8 or control were treated with HIV-1_YU2 (MOI = 0.1) for 8 h and then cell culture medium was changed with fresh medium. The supernatant was collected at the setting time points for p24 ELISA assay. Data were analyzed by unpaired t-test and error bars showed the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001)