Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.
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Technical Advances and Resources|
January 12 2021
A robust platform for expansion and genome editing of primary human natural killer cells
Rih-Sheng Huang
,
Rih-Sheng Huang
Conceptualization, Formal analysis, Methodology, Software, Validation, Visualization, Writing - original draft
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
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Min-Chi Lai
,
Min-Chi Lai
Data curation, Investigation, Project administration, Supervision, Validation, Visualization
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
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Hsin-An Shih
,
Hsin-An Shih
Formal analysis, Investigation, Methodology
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Search for other works by this author on:
Steven Lin
Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Correspondence to Steven Lin: stevenlin@gate.sinica.edu.tw
Search for other works by this author on:
Rih-Sheng Huang
Conceptualization, Formal analysis, Methodology, Software, Validation, Visualization, Writing - original draft
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Min-Chi Lai
Data curation, Investigation, Project administration, Supervision, Validation, Visualization
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Hsin-An Shih
Formal analysis, Investigation, Methodology
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Steven Lin
Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Correspondence to Steven Lin: stevenlin@gate.sinica.edu.tw
Disclosures: The authors declare no competing interests exist.
Received:
July 19 2020
Revision Received:
October 08 2020
Accepted:
November 24 2020
Online Issn: 1540-9538
Print Issn: 0022-1007
Funding:
Academia Sinica
(NO AWARD)
© 2021 Huang et al.
2021
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Exp Med (2021) 218 (3): e20201529.
Article history
Received:
July 19 2020
Revision Received:
October 08 2020
Accepted:
November 24 2020
Citation
Rih-Sheng Huang, Min-Chi Lai, Hsin-An Shih, Steven Lin; A robust platform for expansion and genome editing of primary human natural killer cells. J Exp Med 1 March 2021; 218 (3): e20201529. doi: https://doi.org/10.1084/jem.20201529
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