Figure 2. Elimination of integrated HIV-1 DNA from the host T cell genome by Cas9/gRNAs targeting viral LTRs.

(A) DNA analysis shows 497- and 504-nucleotide amplicons detected, corresponding respectively to the HIV-1 LTRs in control cells and in cells co-expressing Cas9 and gRNAs. Positions of the amplicons corresponding to the RRE and β-actin are shown. (B) Nucleotide composition of the amplified LTR DNA from CRISPR/Cas9-treated cells along with the positions of primers used for PCR amplification of the various regions of the viral genome. Integration of the 7-nucleotide InDel mutation after removal of the viral DNA fragment positioned between the B-motif of the 5′ and 3′ LTRs is shown. The seed sequence for gRNA B is highlighted in black. (C,D) The sites of HIV-1 integration in Chromosome 1 (Panel C) and Chromosome 16 (Panel D) are shown. In each panel, results of DNA analysis of the PCR product amplified by the specific primers (P1 and P2) derived from the cellular genes interrupted by viral DNA insertions are shown. Diagrams of each chromosome containing full-length integrated HIV-1 DNA before CRISPR/Cas9 treatment and the residual LTR DNA sequence after Cas9/gRNAs treatment are depicted, based on Sanger sequencing of the major DNA fragments seen on agarose gel. The asterisks in Panels C and D point to the minor DNA bands indicating the complete removal of viral DNA when either A or B targets within the 5′ or 3′ LTRs were used.