<p>a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear DNA cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two CEM-T4 clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.</p
