Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins.

Frontiers in Immunology
Rih-Sheng HuangSteven Lin

Abstract

Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhan...Continue Reading

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Citations

Sep 26, 2020·Frontiers in Pharmacology·Saeede Soleimanian, Ramin Yaghobi
Jan 5, 2021·Frontiers in Immunology·Simone MantessoLucia Kučerová
Jan 19, 2021·Clinical & Translational Immunology·Luke RigganTimothy E O'Sullivan
Mar 23, 2021·Frontiers in Immunology·Darel Martínez BedoyaDenis Migliorini
Sep 28, 2021·The CRISPR Journal·Chen-Tran HsuChoun-Sea Lin
Sep 17, 2021·Current Protocols·Rih-Sheng HuangSteven Lin
Nov 28, 2021·Nature Communications·Emmanouela TsagkarakiMichael P Czech

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Methods Mentioned

BETA
leukapheresis
genetic modifications
transfection
flow
fluorescence-activated cell sorting
FACS
flow cytometry
in vitro transcription
PCR
Assay

Software Mentioned

lllumina
Sol2
Trimmomatic
CRISPresso2
BclToFastq
Inference of Edits ( ICE )
NEBuilder
Inference of Edit ( ICE )

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