Figure 1

Expression of Cas9 by the HIV-1 LTR promoter is stimulated by Tat leading to cleavage of the viral promoter in the presence of gRNA.

(a) Schematic presentation of the full-length HIV-1 LTR and the various regulatory motifs within the enhancer and core regions and the partial Gag gene. The extent of LTR deletion mutants that are created for expression of Cas9 is depicted. The positions of the gRNA target sequence and their distance from each other is shown. (b) Co-transfection of TZM-bl cells with pX260-LTR-Cas9 containing the full-length LTR (−454/+66) or its various mutants (−120/+66 or −80/+66) along with a plasmid expressing Tat (pCMV-Tat) increased the level of Cas9 production as tested by Western blot (top panel). Expression of housekeeping α-tubulin (middle panel) and Tat (bottom panel) are shown. (c) Infection of TZM-bl cells with adenovirus expressing GFP or Tat followed by transduction with lentivirus expressing Cas9 by the LTR_−80/+66 promoter and gRNAs A/B by the U6 promoter at three different MOI of 2, 4 and 8 led to cleavage of the integrated HIV-1 LTR promoter DNA sequence and the appearance of a 205 bp DNA fragment in the TZM-bl cells (as tested by PCR and DNA gel analysis). (d) SDS-PAGE illustrating the level of Cas9, α-tubulin and Tat protein expressed in TZM-bl cells as described in Panel C. E. Luciferase assay illustrating transcriptional activity of the integrated HIV-1 LTR in TZM-bl cells after various treatments as described in Panel c.