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Fig. 1 | Retrovirology

Fig. 1

From: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

Fig. 1

CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the Bsmb1. b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in (b) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1NL4-3 infection. Neg, no HIV-1 infection. f HIV-1NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For (c and e), one representative out of three independent experiments is shown. For (f), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (***P < 0.001; **P < 0.01; *P < 0.05)

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