3.4 Gene Targeting
and Isolation of Gene
Edited Cells by FACS
Sorting
When the CRISPR/Cas9 reagents are ready, you can proceed to
genome editing with CRISPR/Cas9 to generate cell lines that
express an endogenously tagged protein. The strategy detailed
below relies on antibiotic selection combined with fluorescenceactivated cell sorting (FACS) for enrichment in HDR during
genome editing. FACS is an efficient technology which allows to
simultaneously measure the knock-in efficiency by counting fluorescently positive cells and sort isolated cells into 96-well plates. As
cell sorting relies on the expression levels of the fluorescent-tagged
proteins driven by endogenous promoters, isolation of positive
clones can be challenging for proteins expressed at low levels.
3.4.1 Nucleofection
and Antibiotic Selection
1. Proceed as detailed in Subheading 3.2.1 with a master mix
containing 2 μg of the selected guide sgRNA plasmid
(sgXPB), 2 μg of plasmid expressing the SpCas9 protein
(JDS246), and 6 μg of donor plasmid in a total volume lower
than 10 μL.
2. Three days after transfection, replace the culture medium and
add puromycin to a concentration of 5 μg/mL and further
incubate for an additional week. The use of puromycin selection can be avoided if the signal of the fluorescently tagged
protein is sufficiently strong and the proportion of fluorescent
cells is sufficient for FACS sorting.
3.4.2 Isolation of Gene
Edited Cells by FACS
Sorting
Proceed to FACS sorting 7 days after the beginning of the puromycin treatment (or 3 days after nucleofection in absence of antibiotic selection). Use non-transfected cells as negative control for
gating.
1. Prepare 96-well plates containing 100 μL of growth medium in
each well.
2. Wash cells with PBS, add 1 mL of trypsin and transfer the
resuspended cells in a 1.5-mL microcentrifuge tube containing
0.5 mL of medium.
3. Centrifuge the cell suspension for 5 min at 90 Â g at room
temperature, remove the supernatant, resuspend the cells in
1 mL PBS containing 2% FBS, and filter the cells through a
50-μm cell strainer into a sterile flow cytometry tube.
4. Turn on the cytometer, run the cleaning/calibration procedures and load settings to detect GFP. Refer to the user manual
of your instrument for detailed procedures.
5. Run the non-transfected cells to check the cell sorter parameters and establish gates to select single live cells (Fig. 5).
For single cells, plot forward scatter linear (FCS) on the y-axis
and the fluorescent channel (GFP-FL) on the x-axis. Ideally, set
a background threshold where less than 0.1% of the
non-transfected control cells are counted as GFP positive.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
51
and Isolation of Gene
Edited Cells by FACS
Sorting
When the CRISPR/Cas9 reagents are ready, you can proceed to
genome editing with CRISPR/Cas9 to generate cell lines that
express an endogenously tagged protein. The strategy detailed
below relies on antibiotic selection combined with fluorescenceactivated cell sorting (FACS) for enrichment in HDR during
genome editing. FACS is an efficient technology which allows to
simultaneously measure the knock-in efficiency by counting fluorescently positive cells and sort isolated cells into 96-well plates. As
cell sorting relies on the expression levels of the fluorescent-tagged
proteins driven by endogenous promoters, isolation of positive
clones can be challenging for proteins expressed at low levels.
3.4.1 Nucleofection
and Antibiotic Selection
1. Proceed as detailed in Subheading 3.2.1 with a master mix
containing 2 μg of the selected guide sgRNA plasmid
(sgXPB), 2 μg of plasmid expressing the SpCas9 protein
(JDS246), and 6 μg of donor plasmid in a total volume lower
than 10 μL.
2. Three days after transfection, replace the culture medium and
add puromycin to a concentration of 5 μg/mL and further
incubate for an additional week. The use of puromycin selection can be avoided if the signal of the fluorescently tagged
protein is sufficiently strong and the proportion of fluorescent
cells is sufficient for FACS sorting.
3.4.2 Isolation of Gene
Edited Cells by FACS
Sorting
Proceed to FACS sorting 7 days after the beginning of the puromycin treatment (or 3 days after nucleofection in absence of antibiotic selection). Use non-transfected cells as negative control for
gating.
1. Prepare 96-well plates containing 100 μL of growth medium in
each well.
2. Wash cells with PBS, add 1 mL of trypsin and transfer the
resuspended cells in a 1.5-mL microcentrifuge tube containing
0.5 mL of medium.
3. Centrifuge the cell suspension for 5 min at 90 Â g at room
temperature, remove the supernatant, resuspend the cells in
1 mL PBS containing 2% FBS, and filter the cells through a
50-μm cell strainer into a sterile flow cytometry tube.
4. Turn on the cytometer, run the cleaning/calibration procedures and load settings to detect GFP. Refer to the user manual
of your instrument for detailed procedures.
5. Run the non-transfected cells to check the cell sorter parameters and establish gates to select single live cells (Fig. 5).
For single cells, plot forward scatter linear (FCS) on the y-axis
and the fluorescent channel (GFP-FL) on the x-axis. Ideally, set
a background threshold where less than 0.1% of the
non-transfected control cells are counted as GFP positive.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
51