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Immunology and Infection

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

Published: June 14th, 2018

DOI:

10.3791/58237

1Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital, 2Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, 3Center for Clinical and Translational Research, Nationwide Children's Hospital Research Institute

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–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 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 TGFβ. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

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 ....

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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’s Hospital.

1. Human NK Cell Purification and Expansion

  1. Isolate PBMCs from Buffy Coat12.
    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 PB.......

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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.......

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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.......

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

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

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