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

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Human NK Cell Purification and Expansion

1. Isolate peripheral blood mononuclear cells (PBMCs) from Buffy Coat.
   1. Layer 35 mL of buffy coat sample on 15 mL of Ficoll-Paque.
   2. Centrifuge at 400 x g for 20 minutes without brake and collect the PBMC from the interface.
   3. Wash the recovered PBMCs three times by adding at least an equal volume of PBS, centrifuging at 400 x g for 5 minutes, and aspirating the PBC wash. NK cell can be isolated at this stage by RosettesSep.
2. Expand NK cells by stimulating with irradiated 10 x 10⁶ mbIL21-expressing feeder cells at Days 0, 7, and 14. Replace media with fresh RPMI containing 10% FBS, 1% Glutamine, 1% Penicillin Streptomycin, and 100 IU/mL of IL-2 for the entire media volume every other day.

2. gRNA Design and Selection

1. Choose the specific genomic loci to target, using online tools, e.g., NCBI, Ensemble.
   Example.
   Description: Transforming growth factor beta receptor 2 (TGFBRR2) ectodomain
   View record: PF08917
   View InterPro: IPR015013
   Position: 49 – 157 aa
   Targeted Sequence: Exon 4 of TGFBR2 gene (ENSG00000163513)
2. To design the gRNAs, use CRISPR design web tools such as http://crispr.mit.edu and 'Benchling.'
   1. Enter in the DNA sequence chosen in step 2.1. Choose human (hg 19) as a target genome. CRISPR guides (20 nucleotides followed by a protospacer adjacent motif (PAM) sequence: NGG) will be scanned from the sequence entered earlier. It also shows possible off-target matches throughout the selected genome.
   2. Choose the best three gRNAs which have the highest score, based on their on-target and off-target rates. For example, Table 1 shows the designed CRISPR RNAs to target exon 4 of TGFBR2 gene suggested by CRISPR design web tools.
3. Order the CRISPR RNAs as synthetic sequence-specific crRNAs.
4. Order a conserved, transactivating RNA (tracrRNA) to interact through partial homology with the crRNA.

3. Design Deletion Screening Primers

1. Design primers spanning the gRNA cleavage sites for T7E1 mutation assay.
2. Use primers at least 100 bp away from the predicted cleavage site to ensure small insertion-deletion (indels) at the sgRNA target site will appear on 1.5% agarose gel following the mutation assay. Table 2 shows the primers which are used to amplify the TGFBR2 ectodomain.

4. Transduction of Human Primary and Expanded NK Cells

Note: Transduction of Cas9/RNPs elements into NK is done by electroporation using 4D system as follows.

1. Cell Preparation
   1. For primary NK cells, incubate freshly isolated NK cells in Roswell Park Memorial Institute (RPMI) medium in the presence of 100 IU/mL of IL-2 for 4 days and perform the electroporation at Day 5. Replace the media every other day as described earlier and the day before transduction.
   2. For expanded NK cells, stimulate the cells at day 0 with irradiated feeder cells at a ratio of 1:1 and perform the electroporation at Day 5 or 6 or 7. Replace the media every other day as described earlier and the day before transduction.
   3. On the day of electroporation, prepare a T25 flask filled with 8 mL of fresh RPMI containing 100 IU/mL of IL-2 for cells undergoing electroporation and pre-incubate flasks in a humidified 37 °C and 5% CO₂ incubator.
      Note: Thawed cells or cells that have undergone 2nd or 3rd stimulation can be electroporated at any time after their recovery as described.
   4. Take 3 – 4 × 10⁶ cells per condition for 26 µL of transduction mix.
      Note: Very high concentration of NK cells in electroporation solution enhances the transduction rate.
   5. Wash the cells 3 times with PBS to remove all FBS, which commonly contains RNase activity. Spin them down each time at 300 x g for 8 minutes.
      Note: Consider 7 electroporation conditions for Cas/RNPs as single gRNA (gRNA1, gRNA2, gRNA3) and a combination of two gRNAs (gRNA1+gRNA2, gRNA1+gRNA3, gRNA2+gRNA3) and one control with no Cas9/RNPs.
2. Form the crRNA:tracerRNA/complex
   1. Resuspend crRNAs (gRNA1, gRNA2, and gRNA3) and tracerRNA in 1x TE solution to final concentrations of 200 μM. Mix 2.2 μL of each 200 μM gRNA with 200 μM tracerRNA as shown in Table 3.
   2. Heat the samples at 95 °C for 5 min and allow to cool on the bench top to room temperature (15 – 25 °C). Store resuspended RNAs and crRNA: tracrRNA/complex at -20 °C for later use.
3. Form the RNP complex
   Note: To save time, form the RNP complex during the washing step 4.1.5.
   1. For single crRNA:tracrRNA duplex reaction, dilute Cas9 endonuclease to 36 μM as shown by the example in Table 4.
   2. For combination transduction of crRNA:tracrRNA duplexes, dilute Cas9 endonuclease to 36 μM as shown by the example in Table 5.
   3. Add Cas9 endonuclease to crRNA:tracrRNA duplexes slowly while swirling pipette tip, over 30 s to 1 minute.
   4. Incubate the mixture at room temperature for 15 – 20 min. If not ready to use the mixture after incubation, keep the mixture on ice until use.
4. Electroporation
   1. Add the entire supplement to the electroporation solution P3 and keep it at room temperature.
   2. Resuspend the cell pellet (3 – 4 × 10⁶ cells from step 4.1.5) in 20 μL of P3 primary 4D electroporation solution. Avoid air bubbles while pipetting.
      Note: The cells should not be left for a long time in P3 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.

Table 1. Three designed gRNAs to target exon 4 of TGFBR2 ectodomain as synthetic crRNA.

Table 2. Primers used to amplify the TGFBR2 ectodomain gene

Table 3. Form the crRNA:tracrRNA/complex using 200 µM RNAs

Table 4. For single crRNA:tracrRNA duplex reaction, dilute Cas9 endonuclease to 36 µM.

Table 5. For combination transduction of crRNA:tracrRNA duplexes dilute Cas9 endonuclease to 36 µM.