Figure 5.

Multiplexed tat/rev-targeting all-in-one vectors and their HIV-1 inhibition properties. (a) Schematics of vectors harboring three tat-targeting (lentiCRISPR-tat-3), three rev-targeting (lentiCRISPR-rev-3), and six tat- and rev-targeting gRNAs (lentiCRISPR-tatrev-6). Multiplexed gRNAs from px330A were cloned into plentiCRISPRv2. (b) Functional assay of the developed multiplexed vectors; three plasmid clones for each tat-3, rev-3, and tatrev-6 were tested. The transfections were done for three days into transformant 293T cells, which stably express Tat and Rev proteins. The cells were lysed and immunoblotted with anti-FLAG and anti-β-actin as a loading control. The representative blot images and densitometry graph of FLAG-Tat and Rev-FLAG expression standardized to control are shown. (c) gRNA-Cas9-transduced T cells were infected with HIV-1_NL4-3 equivalent to 200 pg of p24 and cultured for 45 days. The assay was not performed in WT or Cas9-only cells from 15 dpi onward, as extensive cell deaths were apparent. The supernatants were collected at selected time points (5, 10, 15, 20, 30, and 45 dpi) for p24 measurement, and the cell density was determined with trypan blue exclusion every five days. The assays were performed three times with the average values (±SE) shown here. (d) Similar assays were done with an increased titer of HIV_NL4-3: equivalent to 2 ng and 20 ng of p24, with selected gRNA groups. Average values (±SE) of three replicates are shown.