A CRISPR-Cas9 Technique for Gene Editing in T Cells

Take T cells suspended in a medium suitable for electroporation, a process in which electric pulses create temporary pores in the cell membrane.

Add complexes containing a Cas9 protein and a single guide RNA or sgRNA. sgRNA is a small RNA that directs Cas9, a molecular scissor, to a specific location on the DNA for cutting.

Add short single-stranded DNA molecules that assist in transporting the Cas9-sgRNA complexes into the cell.

Transfer this mixture into a cuvette and initiate electroporation by applying brief electric pulses.

This process allows the complexes to enter the cell.

The sgRNA binds to the target site in the T cell DNA, allowing Cas9 to edit the DNA, creating a break in both strands.

This disruption signals the repair proteins to the site.

The repair proteins then reconnect the ends, making the gene non-functional.

Incubate the gene-edited T cells in a suitable medium for downstream assays.

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.