Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

To disrupt granulocyte-macrophage colony-stimulating factor, utilize a guide RNA, as described in the manuscript. Incubate transfection reagents at room temperature for 30 minutes. After incubation, add them to the 293T cells that have reached 70% to 90% confluency, and culture the transfected cells at 37 degrees Celsius, 5% CO₂ to produce lentivirus.

At 24 and 48 hours, harvest, and concentrate virus-containing supernatant by ultracentrifugation in 50-milliliter ultracentrifuge tubes, and freeze at minus 80 degrees Celsius for future use. Then, on day one, gently resuspend the T cells to break up rosettes.

In a BSL-2 plus approved laboratory, to generate CAR T cells, add CAR19 lentivirus and GM-CSF-targeting CRISPR lentivirus to the stimulated T cells. On day three and day five, for successfully transduced lenti-CRISPR-edited T cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of 1 microgram of puromycin per milliliter.