A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9

Take a mixture of a CRISPR RNA, or crRNA, and a trans-activating crRNA or tracrRNA.

Heat the mixture and allow it to cool, facilitating the base pairing of tracrRNA with crRNA to form a guide RNA, or gRNA.

Add molecular scissors called Cas9 enzymes that bind to the gRNA, creating complexes.

Add the Cas9-gRNA complexes to Natural Killer or NK cells suspended in an electroporation solution.

Introduce single-stranded DNA molecules that aid in transporting Cas9-gRNA complexes.

Transfer the mixture into a cuvette and perform electroporation, creating temporary pores in the membrane for the complexes to enter the cell.

The gRNA binds to the target site in the genome, allowing Cas9 to cleave the DNA.

Repair proteins bind at the break site and join the DNA, often causing base pair insertion or deletion errors. The resulting modification disrupts the target gene, making it non-functional.

Transfer the genetically modified cells to a suitable medium for further analysis.

On the day of electroporation, fill one T25 flask with 8 milliliters of fresh culture medium supplemented with 100 IU per milliliter of IL-2 per experimental cell condition, and place the flasks in a humidified 37 degrees Ce