Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

Begin with a suspension of T cells and treat them with CAR lentiviruses carrying a gene for the chimeric antigen receptor or CAR and CRISPR lentiviruses.

The CRISPR lentivirus contains genes for the guide RNA and Cas9 nuclease as well as an antibiotic resistance gene.

Both the CAR and CRISPR lentiviruses interact with T cells and release their RNA.

These RNAs are reverse-transcribed and converted into corresponding DNA, which integrates into the T cell DNA.

This enables the synthesis of CAR proteins, guide RNAs, and Cas9 proteins.

The guide RNA forms a complex with Cas9, which specifically recognizes and binds to the target gene on the T cell DNA.

Meanwhile, Cas9 cleaves the DNA, creating a double-stranded break.

This break is repaired via non-homologous end joining, an error-prone repair mechanism, resulting in target gene modification.

This generates genetically modified T cells with chimeric antigen receptors.

Treat the cells with antibiotics to eliminate the unmodified T cells and selectively isolate the genetically modified CAR T cells.

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.