This article describes the process of editing T cell DNA using electroporation and CRISPR technology. The method involves the use of Cas9 protein and single guide RNA (sgRNA) to create targeted DNA breaks, which are then repaired by the cell's machinery, resulting in gene disruption.
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.
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