We acknowledge Brian Tullius for his kind help in editing the manuscript.

Healthy donor buffy coats were obtained as source material from the Central Ohio Region American Red Cross. This research was determined to be exempt research by the Institutional Review Board of Nationwide Children&#8217;s Hospital.
1. Human NK Cell Purification and Expansion
<ol>
	<li>Isolate PBMCs from Buffy Coat12.
	<ol>
		<li>Layer 35 mL of buffy coat sample on 15 mL of Ficoll-Paque.</li>
		<li>Centrifuge at 400 x g for 20 minutes without brake and collect the PB...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+delivery+of+proteins+enables+efficient+protein-based+genome+editing+in+vitro+and+in+vivo.">Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.</a> Nature Biotechnology. 33 (1), 73-80 (2015).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+delivery+of+genome-editing+proteins+using+bioreducible+lipid+nanoparticles.">Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.</a> PNAS. 113 (11), 2868-2873 (2016).</li><li>Liang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+DNA-free+genome+editing+of+bread+wheat+using+CRISPR/Cas9+ribonucleoprotein+complexes.">Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.</a> Nature Communications. 8, 14261 (2017).</li><li>DeWitt, M. A., Corn, J. E., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+via+delivery+of+Cas9+ribonucleoprotein.">Genome editing via delivery of Cas9 ribonucleoprotein.</a> Methods. , 9-15 (2017).</li><li>Rezvani, K., Rouce, R., Liu, E., Shpall, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Natural+Killer+Cells+for+Cancer+Immunotherapy.">Engineering Natural Killer Cells for Cancer Immunotherapy.</a> Molecular Therapy. 25 (8), 1769-1781 (2017).</li><li>Sutlu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells:+implications+for+gene+therapy.">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Human Gene Therapy. 23 (10), 1090-1100 (2012).</li><li>Viel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+inhibits+the+activation+and+functions+of+NK+cells+by+repressing+the+mTOR+pathway.">TGF-beta inhibits the activation and functions of NK cells by repressing the mTOR pathway.</a> Science Signaling. 9 (415), (2016).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> JoVE. (48), (2011).</li><li>Lee, D. A., Verneris, M. R., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition,+preparation,+and+functional+assessment+of+human+NK+cells+for+adoptive+immunotherapy.">Acquisition, preparation, and functional assessment of human NK cells for adoptive immunotherapy.</a> Methods in Molecular Biology. , 61-77 (2010).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5 (3), 407-415 (2015).</li><li>Eyquem, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+CAR+to+the+TRAC+locus+with+CRISPR/Cas9+enhances+tumour+rejection.">Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.</a> Nature. 543 (7643), 113-117 (2017).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+highly+efficient+mammalian+cell+engineering+via+Cas9+protein+transfection.">Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.</a> Journal of Biotechnology. 208, 44-53 (2015).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Chmielecki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Profiling+of+a+Large+Set+of+Diverse+Pediatric+Cancers+Identifies+Known+and+Novel+Mutations+across+Tumor+Spectra.">Genomic Profiling of a Large Set of Diverse Pediatric Cancers Identifies Known and Novel Mutations across Tumor Spectra.</a> Cancer Research. 77 (2), 509-519 (2017).</li><li>Schultz, L. M., Majzner, R., Davis, K. L., Mackall, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+immunotherapy+for+pediatric+solid+tumors.">New developments in immunotherapy for pediatric solid tumors.</a> Current Opinion in Pediatrics. 30 (1), 30-39 (2018).</li></ol>

DAL serves/has served as a consultant for Courier Therapeutics, Obsidian Therapeutics, Intellia Therapeutics, Merck Research Laboratories, and Miltenyi Biotec, and has equity/Leadership in CytoSen Therapeutics.

DNA-dependent modification of NK cells has been challenging4,9. We, therefore, introduced directly a synthetically preformed ribonucleoprotein (RNPs) complex and Cas9 protein as purified protein into primary and expanded NK cells8. This method allowed us to eliminate capping, tailing, and other transcriptional and translational processes started by RNA polymerase II, which may cause NK cell apoptosis associated with DNA-dependent transduct...

Cancer immunotherapy has been advanced in recent years. Genetically-modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells successfully deployed in cancer immunotherapy. These cells were recently approved by the FDA for treatment against CD19 + B cell malignancies, but success has so far been limited to diseases bearing a few targetable antigens, and targeting such limited antigenic repertoires is prone to failure by immune escape. Furthermore, CAR T cells have been focused on the use of autologous T cells because of the risk of graft-versus-host disease caused by allogeneic T cells. In contrast, NK cells are able to kill tumor targets in an antigen-independent manner and do not cause GvHD, which makes them a good candidate for cancer immunotherapy6,7,8,9.
CRISPR/Cas9 technology has been used recently in engineering immune cells, but genetically reprogramming NK cells with plasmids has always been challenging. This has been due to difficulties in transgene delivery in a DNA dependent manner such as lentiviral and retroviral transduction causing substantial procedure-associated NK cell apoptosis and the limited production of genetically engineered NK cells4,9.
Many innate immune cells express high levels of receptors for pathogen-associated molecular patterns such as Retinoic acid-inducible gene I (RIG-I), which enable heightened recognition of foreign DNA. Suppression of these pathways has enabled higher transduction efficiency in NK cells when using DNA-based methods for genetic modification10.
We describe here the method for using a DNA-free genome editing of primary and expanded human NK cells utilizing Cas9 ribonucleoprotein complexes (Cas9/RNPs). Cas9/RNPs is composed of three components, recombinant Cas9 protein complexed with synthetic single-guide RNA comprised of a complexed crRNA and tracerRNA. These Cas9/RNPs are capable of cleaving genomic targets with higher efficiency as compared to foreign DNA-dependent approaches due to their delivery as functional complexes. Additionally, rapid clearance of Cas9/RNPs from the cells may reduce the off-target effects such as induction of apoptosis. Thus, they can be used to generate knock-outs, or knock-ins when combined with DNA for homologous recombination6,7. We showed that electroporation of Cas9/RNPs is an easy and relatively efficient method that overcomes the previous constraints of genetic modification in NK cells.
TGF&#946; is a major immunosuppressive cytokine, which inhibits the activation and functions of NK cells. It has been suggested that targeting the TGF&#946; pathway can increase immune cell functions. We targeted the region encoding TGBR2 ectodomain which binds TGF&#946;11. The representative results show a significant decrease in the level of mRNA expression of this gene and further demonstrate that the modified NK cells become resistant to TGF&#946;. In addition, the modified cells retain viability and proliferative potential, as they are able to be expanded post-electroporation using irradiated feeder cells. Therefore, the following method is a promising approach to genetically manipulate NK cells for any further clinical or research purposes.

<table>
	<tbody>
		<tr>
			<td>RosetteSep&#8482; Human NK Cell Enrichment Cocktail</td>
			<td>STEMCELL Technologies</td>
			<td>15065</td>
			<td>The RosetteSep&#8482; Human NK Cell Enrichment Cocktail is designed to isolate NK cells from whole blood by negative selection.</td>
		</tr>
		<tr>
			<td>Ficoll-Paque&#174; PLUS</td>
			<td>GE Healthcare - Life Sciences</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 tracrRNA</td>
			<td>Integrated DNA Technologies</td>
			<td>1072532</td>
			<td>TracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme</td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 crRNA</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Synthetically produced as Alt-R&#174; CRISPR-Cas9 crRNA, based on the sequence of designed gRNA targeting the gene of intrest</td>
		</tr>
		<tr>
			<td>Alt-R&#174; Genome Editing Detection Kit</td>
			<td>Integrated DNA Technologies</td>
			<td>1075931</td>
			<td>Each kit contains T7EI endonuclease, T7EI reaction buffer, and T7EI assay controls.</td>
		</tr>
		<tr>
			<td>Platinum&#174; Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen</td>
			<td>11304-011</td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofector&#8482; System</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-2 Protein</td>
			<td>Novartis</td>
			<td>65483-0116-07</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector&#8482; X Kit</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td>Contains pmaxGFP&#8482; Vector, Nucleofector&#8482; Solution, Supplement, 16-well Nucleocuvette&#8482; Strips</td>
		</tr>
		<tr>
			<td>Non-targeting: Custom siRNA, Standard 0.05 2mol ON-TARGETplus</td>
			<td>Dharmacon</td>
			<td>CTM-360019</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; S.p. Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1074181</td>
			<td>Cas9 Nuclease</td>
		</tr>
		<tr>
			<td>DNeasy&#174; Blood &#38; Tissue Handbook</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>ThermoFisher</td>
			<td>C3099</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;</td>
			<td>Biolegend</td>
			<td>580706</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 Control Kit, Human</td>
			<td>Integrated DNA Technologies</td>
			<td>1072554</td>
			<td>Includes tracrRNA, HPRT positive control crRNA, negative control crRNA#1, HPRT Primer Mix, and Nuclease-Free Duplex Buffer.</td>
		</tr>
		<tr>
			<td>IDTE pH 7.5 (1X TE Solution)</td>
			<td>Integrated DNA Technologies</td>
			<td>11-01-02-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; Cas9 Electroporation Enhancer</td>
			<td>Integrated DNA Technologies</td>
			<td>1075915</td>
			<td>Cas9 Electroporation Enhancer</td>
		</tr>
	</tbody>
</table>

generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

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 &#38; Figure 2...

Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

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

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

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.

generation-knock-out-primary-expanded-human-nk-cells-using-cas9

Research

JoVE Journal

Immunology and Infection

12.3K ビュー.  Nationwide Children's Hospital. 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...