Fig. 4

Generation of a stable cell clone with TK-SR39 integration and CCR5 KO. a 293T cells were transfected to generate CCR5gRNA CRISPR/Cas9 Tat NIL particles. TZM-TK-SR39 cells were transduced with the NIL lentivirus and the cells monitored for GFP expression and CCR5 down regulation. CCR5 negative cells were sorted 3 times to generate sort 1–3 (S1, S2, S3) followed by single cell cloning. b Flow cytometry analysis of (b). The TZM-TK-SR39 cells transduced with control gRNA packaged lentivirus or CCR5 gRNA lentivirus were stained for CCR5 expression prior to cell sorting. c The sorted cells (Sort 1–3) were assayed for surface CCR5 expression by flow cytometry. d Sort-3 cells were further subjected to single cell cloning and several potential clones screened for CCR5 downregulation. The selected clone H7 was stained for CCR5 and compared to the parental Sort-3 cells