Chapter 15
CRISPR/Cas9-Mediated GFP Reporter Knock-in in K562
and Raji Cell Lines for Tracking Immune Cell Killing Assay
Nontaphat Thongsin and Methichit Wattanapanitch
Abstract
Cell-mediated cytotoxicity plays an important role in several fundamental immunological processes and is
crucial for biological evaluation in in vitro studies. In order to determine the immunological activities of the
cells, an assay should be safe, reproducible, and cost-effective. Here, we present a simple and cost-effective
approach for evaluation of natural killer (NK) cell-mediated cytotoxicity by generating a CRISPR/Cas9mediated GFP reporter knock-in in the target cell line, K562, and the non-target cell line, Raji, using a
plasmid-based transfection method. The GFP
+ target cells facilitate tracking of the immune cell killing assay,
which avoids the need for multiple cell labeling with fluorescent dyes. Our approach is also applicable to the
genome editing of other target cell types for functional analysis of effector cells.
Key words CRISPR/Cas9, GFP knock-in, K562, Raji, Immune cell killing, NK-92 cells, Clonal
selection
1 Introduction
Cell-mediated cytotoxicity is the most important effector pathway
of immunological response against intracellular pathogens or
tumor cells [1]. This process is considered a hallmark of CD8
+
cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function
[2, 3]. In addition, the mechanism is crucial for the biological
evaluation of effector and/or target cell response in vitro
[4]. There are two major contact-dependent cytotoxic pathways
involved in the elimination of the target cells. The first pathway
involves secretion of a pore-forming protein called perforin, and the
pro-apoptotic serine protease family called granzyme B, which
activates caspases. The second pathway involves the production of
extrinsic apoptosis signaling proteins such as TNF-α, FasL, or
TRAIL, which trigger apoptosis of the target cells [5, 6].
In the past decades, the radioactive chromium (
51 Cr)-release
assay was the first quantification method for cell-mediated cytotoxicity, as described by Brunner et al. [7]. This assay is based on the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
213
CRISPR/Cas9-Mediated GFP Reporter Knock-in in K562
and Raji Cell Lines for Tracking Immune Cell Killing Assay
Nontaphat Thongsin and Methichit Wattanapanitch
Abstract
Cell-mediated cytotoxicity plays an important role in several fundamental immunological processes and is
crucial for biological evaluation in in vitro studies. In order to determine the immunological activities of the
cells, an assay should be safe, reproducible, and cost-effective. Here, we present a simple and cost-effective
approach for evaluation of natural killer (NK) cell-mediated cytotoxicity by generating a CRISPR/Cas9mediated GFP reporter knock-in in the target cell line, K562, and the non-target cell line, Raji, using a
plasmid-based transfection method. The GFP
+ target cells facilitate tracking of the immune cell killing assay,
which avoids the need for multiple cell labeling with fluorescent dyes. Our approach is also applicable to the
genome editing of other target cell types for functional analysis of effector cells.
Key words CRISPR/Cas9, GFP knock-in, K562, Raji, Immune cell killing, NK-92 cells, Clonal
selection
1 Introduction
Cell-mediated cytotoxicity is the most important effector pathway
of immunological response against intracellular pathogens or
tumor cells [1]. This process is considered a hallmark of CD8
+
cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function
[2, 3]. In addition, the mechanism is crucial for the biological
evaluation of effector and/or target cell response in vitro
[4]. There are two major contact-dependent cytotoxic pathways
involved in the elimination of the target cells. The first pathway
involves secretion of a pore-forming protein called perforin, and the
pro-apoptotic serine protease family called granzyme B, which
activates caspases. The second pathway involves the production of
extrinsic apoptosis signaling proteins such as TNF-α, FasL, or
TRAIL, which trigger apoptosis of the target cells [5, 6].
In the past decades, the radioactive chromium (
51 Cr)-release
assay was the first quantification method for cell-mediated cytotoxicity, as described by Brunner et al. [7]. This assay is based on the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
213