Nucleofection

1. Add the entire supplement to the electroporation solution P3 and keep it at room temperature.

2. Resuspend the cell pellet (3 - 4 × 1,0E+6 cells from step 4.1.5) in 20 µL of P3 primary 4D electroporation solution.

3. Immediately add 5 µL of RNP complex (Step 4.3) to the cell suspension.

4. Add 1 µL of 100 µM Cas9 electroporation enhancer to the Cas9/RNPs/cell mix.

5. Transfer Cas9/RNPs/cell mix into 20 µL electroporation strips.

6. Gently tap the strips to make sure that the sample covers the bottom of the strips.

7. Start 4D electroporation system and choose the EN-138 program.

Post Transduction

1. Let the cells rest for 3 minutes in the strips.

2. Add 80 µL of the pre-equilibrated culture media to the cuvette and gently transfer the sample into flasks.

3. 48 hours after transduction, extract genomic DNA from 5 × 105 cells for the gene deletions screening

...

Electroporation Efficiency:

To optimize electroporation of Cas9/RNPs, we tested 16 different programs with transduction of GFP non-targeting siRNA and DNA plasmid into NK cells. Flow cytometry assay showed that the EN-138 had the highest percentage of cell viability and transduction efficiency (35% live GFP positive cells) for both particles (Figure 1 & Figure 2). Interestingly, the efficiency of using this program for Cas9/RNPs electroporation was higher as we saw 60% reduction in TGFBR2 mRNA expression level (Figure 5). Additionally, the genetically modified NK cells could be grown and expanded for 30 days and cryopreserved (data not shown).

Lonza summary:

Method paper describing efficient gene knockouts using CRISPR Cas9 RNPs and the 4D Nucleofector - X unit in human primary NK cells derived from PBMCs, then the proficient growth and expansion of the modified NK cells for up to 1-month time