2. Academic Validation
  3. Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

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

  • J Vis Exp. 2018 Jun 14:(136):58237. doi: 10.3791/58237.
Meisam Naeimi Kararoudi 1,  Hamid Dolatshad 2,  Prashant Trikha 1,  Syed-Rehan A Hussain 3,  Ezgi Elmas 1,  Jennifer A Foltz 1,  Jena E Moseman 1,  Aarohi Thakkar 1,  Robin J Nakkula 1,  Margaret Lamb 1,  Nitin Chakravarti 1,  K John McLaughlin 3,  Dean A Lee 4
Affiliations

Affiliations

  • 1 Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital.
  • 2 Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford.
  • 3 Center for Clinical and Translational Research, Nationwide Children's Hospital Research Institute.
  • 4 Center for Childhood Cancer and Blood Disease, Nationwide Children's Hospital; Dean.lee@nationwidechildrens.org.
PMID: 29985369 DOI: 10.3791/58237
Abstract

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.

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