Fig. 1
Efficient disruption of CCR5 in TZM-b1 cells by AAV-CRISPR/SaCas9 defenses HIV-1 infection. a The diagram of AAV-CRISPR-SaCas9 and Lenti-CRISPR-SaCas9 vector composition and insert sites of CCR5-sgRNA. b T7E1 assay analysis of disruption efficiency in TZM-bl cells transfected with AAV-CRISPR/SaCas9-sgRNA-#6, #8 or control. The indel percentage was calculated using Image J software. c Flow cytometry detection of CCR5 expression on cell surface. The TZM-bl cells transfected with AAV-SaCas9/sgRNA-#6, #8 or control were stained with APC-conjugated CCR5 antibody and analyzed by flow cytometry. The unstained control-transfected TZM-bl cells and stained control-transfected TZM-bl cells were treated as negative and positive controls respectively. The data showed on the top of each peak were the percentage of CCR5 negative and positive cells. d DNA sequences of CCR5 target sites in the TZM-bl cells mediated by sgRNA-#6 and #8. PCR products amplification from genomic DNA were cloned into T-vector and performed Sanger sequencing. The red sequences indicate the PAM sequences; the blue sequences are marked as the targeted sequences; the green bases in the targeted sequences are mutations; insertions and deletions are indicated with (+) and (−) respectively. N/N shows the ratio of mutations or wild type (WT) in the all sequenced clones. e Disruption of CCR5 in TZM-bl cells resistance to HIV-1 infection. TZM-bl cells edited by sgRNA-#6, #8 or control were infected with R5-tropic HIV-1YU2 (MOI = 0.5) and analyzed by luciferase reporter assay. Data were analyzed by unpaired t-test and error bars showed the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001)