Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

This method can help answer key questions in the NK cell biology field about the role and function of specific genes and pathways that regulate effector cell function. The main advantage of this technique is its high success rate compared to DNA-dependent approaches facilitating a highly efficient knockout of one of the most clinically relevant genes in NK cell therapy. The implications of this technique extend toward the immunotherapy of solid tumors and brain tumors that express TGF-beta as a means of immune escape.

For primary NK cell transduction, incubate three times 10 to the five freshly isolated NK cells per milliliter of RPMI medium supplemented with 100 IU per milliliter of IL2 per T75 flask for four days replacing the medium every other day and the day before transduction. For expanded NK cell transduction, stimulate the primary NK cells at day zero with a radiated membrane-bound IL21-expressing K562 feeder cells at a one-to-one ratio replacing the medium every other day and the day before transduction. On the day of electroporation, fill one T25 flask with eight milliliters of fresh culture medium supplemented with 100 IU per milliliter of IL2 per experimental cell condition and place the flasks in a humidified 37 degree Celsius and 5%CO2 incubator.

While the medium is equilibrating, add three to four times 10 to the sixth 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 tracrRNA to 200 micromolar final concentrations.

Mix 2.2 microliters of each CRISPR RNA with one 200 micromolar volume of tracrRNA per sample and heat the samples at 95 degrees Celsius for five minutes. Then allow the samples to cool to room temperature and store the CRISPR RNA/tracrRNA complexes at negative 20 degrees Celsius until their use. To form the ribonucleoprotein complexes, add Cas9 endonuclease to the appropriate corresponding CRISPR RNA/tracrRNA 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 five microliters of each ribonucleoprotein complex to the appropriate cell suspension and add one 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 EN138 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.

After 48 hours of culture, extract the genomic DNA from five times 10 to the five transduced cells for gene deletion screening according to standard protocols. Then stimulate the rest of the transduced cells with fresh membrane-bound IL21-expressing feeder cells at a one-to-one ratio and extract the RNA after five days of co-culture according to standard protocols for gene expression analysis by quantitative PCR. Flow cytometry analysis reveals that the EN138 program results in the highest percentage of cell viability and transduction efficiency of the available Nucleofector system programs.

For example, using the commercially-provided guide RNAs, human HPRT1 is successfully knocked out in expanded human NK cells. In addition, Cas9 ribonucleoproteins containing guide RNA2, guide RNA1 plus guide RNA2 and guide RNA3 exhibit a successful TGF-beta receptor two ectodomain gene knockout while guide RNA1 alone does not induce any T7E1 detectable insertion deletions. Indeed, using the EN138 program for Cas9 ribonucleoprotein electroporation results in a 60%reduction in TGF-beta receptor two mRNA expression in cells transduced via this program.

Further, co-culture of the transduced cells with TGF-beta and a human primary medulloblastoma cell line does not significantly impact NK cell cytotoxicity compared to control non-TGF-beta-treated transduced cells, confronting a corresponding lack of TGF-beta receptor two function in the Cas9 ribonucleoprotein modified cells at the protein level. Once mastered, this technique can be completed in five hours if it is performed properly. While attempting this procedure, it is important to remember to design several different guide RNAs to find the best gene editing result.

Following this procedure, other methods like DNA sequencing or the T7E1 mutation assay can be performed to answer additional questions about the efficiency of gene editing or how to determine potential off-target genetic mutations. After its development, this technique paved the way for researchers in the field of cancer immunotherapy to explore the potential for using gene edited NK cells in cancer treatment. After watching this video, you should have a good understanding of how to perform gene editing of primary human NK cells.

For more information on NK cell expansion after this process, please see our earlier Jove article. Don't forget that working with human blood can be extremely hazardous and that universal health and safety precautions should always be taken while performing this procedure.