Figure 4.

Positive selection for CXCR4 knockout cells by HIV-1_NL4-3 infection in hu-PBMCs

(A) Schematic of the timeline of building hu-PBMC mouse model and HIV infection by using mixed human primary PBMCs with CXCR4 CRISPR-modified CD4⁺ T cells. (B) Cell surface CXCR4 co-receptor knockout in CD4⁺ T cells after MaxCyte electroporation of CXCR4 guide RNAs and Cas9 RNPs. Cells were fixed in 2% formaldehyde and analyzed by flow cytometry 48 h after transfection. (C) Surveyor assay detection of the allelic disruption of CXCR4 gene in the CXCR4 CRISPR-modified cells. (D–F) Flow cytometry analysis of human CD3⁺ T cells in mice whole blood after transplantation by using CXCR4 CRISPR-modified or unmodified cells (control). (G) qRT-PCR was performed using plasma from hu-PBMC mice. Mice whole blood was collected by retro-orbital bleeding 2 weeks after HIV-1_NL4-3 infection (6 weeks after transplantation). Data were presented by comparing two groups of mice that were transplanted by using the control and CXCR4 CRISPR-modified cells.