release of the radioactive probe from the pre-labeled lysed target
cells to the culture medium. This assay is considered as the standard
method for cell-mediated cytotoxicity and still widely used. However, high spontaneous release of labelled51 Cr from the target cells
and risks of radioisotope handling are the main drawback of this
assay [8].
In order to avoid difficulties in using the radioisotope, enzymelink immunospot assay (ELISpot) and flow cytometric assay have
been developed. Cell-mediated cytotoxicity using ELISpot is based
on the quantification of locally secreted cytokines captured by
antibody-coated vessels. ELISpot provides more sensitivity and
specificity than the
51
Cr release assay and circumvents the issues
that occurred during cell labeling and spontaneous
51 Cr leakage.
However, ELISpot does not provide a direct measurement of
the target cell lysis and cytotoxicity mediated by FasL pathway
[9, 10].
To overcome these limitations, numerous flow cytometric
approaches have been applied for insight measurement of effector
cells mediated target cell killing. These approaches include evaluation of caspase activity, annexin V binding apoptotic cells, uptake of
propidium iodide (PI), or 7-amino-actinomycin D (7-AAD) by
dying target cells [9]. However, to differentiate between the effector and target cell populations, additional cell labeling with fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE)
[11, 12] or the fluorescent protein such as green fluorescence
protein (GFP) fusion tag [13] is required to facilitate the determination of effector and target cell populations.
Here, we use CRISPR/Cas9 genome editing technique to
generate target cells, which stably express GFP. CRISPR/Cas9
system is a recent genome editing tool, which can precisely modify
the target DNA sequence. The CRISPR/Cas9 component is composed of Cas9 protein, a CRISPR RNA (crRNA) of 20 nucleotides,
which binds to the complementary target DNA, and a non-coding
trans-activating CRISPR RNA (tracrRNA). The crRNA and
tracrRNA can be fused together to form a single guide RNA
(sgRNA) [14].
Interaction of sgRNA and target DNA facilitates the activation
of the catalytic domain of Cas9 nuclease and results in a doublestranded break (DSB) at three nucleotides upstream of the
5
0 -NGG-3
0 (PAM) site [15–17]. In response to DSB, the cells
employ two endogenous repair mechanisms, either non-homologous end joining (NHEJ) or homology-directed repair (HDR)
pathway. The NHEJ pathway repairs the DNA damage by ligating
the broken ends of the DNA strand, independent of sequence
homology. This repair mechanism is, therefore, error-prone, causing base insertions or deletions (indels) at the lesion site. In contrast, the HDR pathway employs the existing DNA molecule with
the sequence homology to the region around the DSB as a template
214
Nontaphat Thongsin and Methichit Wattanapanitch
cells to the culture medium. This assay is considered as the standard
method for cell-mediated cytotoxicity and still widely used. However, high spontaneous release of labelled51 Cr from the target cells
and risks of radioisotope handling are the main drawback of this
assay [8].
In order to avoid difficulties in using the radioisotope, enzymelink immunospot assay (ELISpot) and flow cytometric assay have
been developed. Cell-mediated cytotoxicity using ELISpot is based
on the quantification of locally secreted cytokines captured by
antibody-coated vessels. ELISpot provides more sensitivity and
specificity than the
51
Cr release assay and circumvents the issues
that occurred during cell labeling and spontaneous
51 Cr leakage.
However, ELISpot does not provide a direct measurement of
the target cell lysis and cytotoxicity mediated by FasL pathway
[9, 10].
To overcome these limitations, numerous flow cytometric
approaches have been applied for insight measurement of effector
cells mediated target cell killing. These approaches include evaluation of caspase activity, annexin V binding apoptotic cells, uptake of
propidium iodide (PI), or 7-amino-actinomycin D (7-AAD) by
dying target cells [9]. However, to differentiate between the effector and target cell populations, additional cell labeling with fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE)
[11, 12] or the fluorescent protein such as green fluorescence
protein (GFP) fusion tag [13] is required to facilitate the determination of effector and target cell populations.
Here, we use CRISPR/Cas9 genome editing technique to
generate target cells, which stably express GFP. CRISPR/Cas9
system is a recent genome editing tool, which can precisely modify
the target DNA sequence. The CRISPR/Cas9 component is composed of Cas9 protein, a CRISPR RNA (crRNA) of 20 nucleotides,
which binds to the complementary target DNA, and a non-coding
trans-activating CRISPR RNA (tracrRNA). The crRNA and
tracrRNA can be fused together to form a single guide RNA
(sgRNA) [14].
Interaction of sgRNA and target DNA facilitates the activation
of the catalytic domain of Cas9 nuclease and results in a doublestranded break (DSB) at three nucleotides upstream of the
5
0 -NGG-3
0 (PAM) site [15–17]. In response to DSB, the cells
employ two endogenous repair mechanisms, either non-homologous end joining (NHEJ) or homology-directed repair (HDR)
pathway. The NHEJ pathway repairs the DNA damage by ligating
the broken ends of the DNA strand, independent of sequence
homology. This repair mechanism is, therefore, error-prone, causing base insertions or deletions (indels) at the lesion site. In contrast, the HDR pathway employs the existing DNA molecule with
the sequence homology to the region around the DSB as a template
214
Nontaphat Thongsin and Methichit Wattanapanitch