Figure 3

Tat stimulation of Cas9 cleaves integrated HIV-1 DNA in T-cells encompassing the HIV-1 reporter at a latent stage.

2D10 cells with integrated copies of LTR_−80/+66-Cas9 gene were transfected with control (pKLV) or pKLV-gRNA A/B along with pCMV or pCMV-Tat plasmids. After 48 hours, the level of various proteins, as depicted, was determined by Western blot (a). The genomic DNA for assessing the state of the integrated HIV-1 DNA was determined by LTR specific PCR and the excision efficiency was determined as a percentage of ratios between truncated vs. full-length amplicon and presented in arbitrary units (AU) 0–0.5 (b). The level of integrated viral promoter reactivation after cleavage was assessed by flow cytometry and the representative scatter plots are shown (c). Red positive, propidium iodide labeled, dead cells were excluded from the analysis.