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

Meisam Naeimi Kararoudi, Hamid Dolatshad, Prashant Trikha, Syed Rehan A. Hussain, Ezgi Elmas, Jennifer A. Foltz, Jena E. Moseman, Aarohi Thakkar, Robin J. Nakkula, Margaret Lamb, Nitin Chakravarti, K. John McLaughlin, Dean A. Lee

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

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.

Original languageEnglish
Article numbere58237
JournalJournal of Visualized Experiments
Volume2018
Issue number136
DOIs
StatePublished - Jun 14 2018

Keywords

  • CRISPR/Cas9
  • Cas9/RNPs
  • GRNAs
  • Human expanded NK cells
  • Human primary NK cells
  • Immunology and Infection
  • Issue 136
  • TGFBR2
  • TGFβ resistant NK cells

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