A CRISPR-Cas9 Technique for Gene Editing in T Cells

Prepare ribonucleoprotein, or RNP complex, by incubating 10 micrograms of Cas9 nuclease with 5 micrograms of single guide RNA for 10 minutes at room temperature. Include a mock control without single guide RNA. Combine the resuspended T cells with the RNP complex and add 4.2 microliters of 4 micromolar electroporation enhancer. Mix well, and transfer into electroporation cuvettes.

Electroplate the cells using pulse code EH-1-11. Then, incubate 5 million cells per milliliter in R-10, supplemented with five nanograms per milliliter human IL-7 and human Il-15 at 30 degrees Celsius for 48 hours in 12-well plates. After the incubation, proceed with T cell activation and expansion.