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 Celsius and 5% CO₂ incubator.

While the medium is equilibrating, add 3 to 4 x 10⁶ cells per condition, per 26 microliters of transduction mix into a conical tube, and pellet the cells by centrifugation. Then, wash the cells three times in 12 milliliters of sterile PBS per wash to remove all of the FBS from the cell suspensions.

While the cells are being washed, resuspend each CRISPR RNA and tracer RNA to 200 micromolar final concentrations. Mix 2.2 microliters of each CRISPR RNA with one 200-micromolar volume of tracer RNA per sample and heat the samples at 95 degrees Celsius for 5 minutes. Then, allow the samples to cool to room temperature, and store the CRISPR RNA - tracer RNA complexes at negative 20 degrees Celsius until their use.

To form the ribonucleoprotein complexes, add Cas9 endonuclease to the appropriate corresponding CRISPR RNA - tracer RNA complex with swirling over 30 to 60 seconds, and incubate the resulting Cas9 ribonucleoprotein complexes at room temperature for 15 to 20 minutes.

After the last wash, add the entire volume of electroporation supplement from the Nucleofector solution P3 kit and resuspend each cell pellet in 20 microliters of the P3 primary 4D-Nucleofector solution, taking care to avoid air bubbles.

Immediately add 5 microliters of each ribonucleoprotein complex to the appropriate cell suspension, and add 1 microliter of 100 micromolar Cas9 electroporation enhancer to the Cas9 ribonucleoprotein complex cell mix. Transfer 26 microliters of the Cas9 ribonucleoprotein cell solution into individual wells of a Nucleocuvette Strip, and gently tap the strip to make sure the samples cover the bottom of each well.

Then, start the 4D-Nucleofector system EN-138 program. At the end of the transduction, let the cells rest for three minutes. Then, add 80 microliters of the pre-equilibrated culture medium to each cuvette, and gently transfer the samples into one flask per condition at 37 degrees Celsius incubation.