A CRISPR-Cas9 Technique for Gene Editing in T Cells
Transcript
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.
