Name: generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins
Uploaded: 2018-06-14T15:00:00
Description: Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

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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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 ...

Research

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