challenging to detect. An attractive alternative consists in delivering
the nuclease with a single strand donor designed to fuse a HiBiT
peptide to the target protein. The abundance of HiBiT-tagged
proteins in transfected cells is proportional to the proportion of
edited gene copies and is readily detected using the Nano-Glo
HiBiT luciferase-based detection system (see ref. 8).
In this section, we detail the delivery of CRISPR/Cas9
reagents by nucleofection (see Note 3) and PCR-based analysis of
the cleavage efficiency of guide RNAs.
3.2.1 Nucleofection
1. Thaw and maintain U2-OS cells using your favorite protocol
(see Note 1). We usually split cells 1:5 every 3–4 days, verify
that the viability is always higher than 95% and limit the number of passages to 20.
2. Two or three days before nucleofection, seed two 75-cm
2 flasks
and incubate at 37
C, 5% CO 2 .
3. When 50–70% confluence is reached, detach the cells with
trypsin, spin-down in a 15-mL tube at 90 g at room temperature for 10 min, remove the supernatant and re-suspend the
cells in PBS to a concentration of 1–3 Â 10
6 cells/mL.
4. Aliquot 10
6 cells into 1.5-mL microcentrifuge tubes (one tube
per guide RNA to be tested plus an additional tube for a
negative control), pellet the cells, discard supernatant
completely, and resuspend cells in Nucleofector
® solution
(100 μL per sample).
5. Prepare the DNA mixes containing 2 μg of guide sgRNA
plasmid (sgXPB) and 2 μg of plasmid expressing the SpCas9
protein (JDS246) in a total volume less than 10 μL.
6. Mix the DNA solution with 100 μL of cell suspension and
transfer to a cuvette. Process the samples quickly to avoid
storing the cells longer than 10 min in Nucleofector
®
Solution V.
7. Insert the cuvette into the Nucleofector
® , select the cell-typespecific program X-001 and press the start button. Using the
provided pipette, immediately remove the sample from the
cuvette and transfer into the six-well plate containing 2 mL of
culture medium.
8. Incubate the six-well plates at 37
C, 5% CO 2 and proceed to
analysis or collect the cells and store the cell pellet at À20
C.
3.2.2 PCR-Based
Analysis of the Cleavage
Efficiency for the sgRNA
CRISPR/Cas9 cleavage is usually assessed by amplifying the target
region by PCR and analyzing the mutation rate in the resulting
product. This can be performed using various assays such as the
polymerase chain reaction (PCR)/restriction enzyme (RE) assay,
T7 endonuclease I (T7EI) assay, Surveyor nuclease assay or with
TIDE [15–18].
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
47
the nuclease with a single strand donor designed to fuse a HiBiT
peptide to the target protein. The abundance of HiBiT-tagged
proteins in transfected cells is proportional to the proportion of
edited gene copies and is readily detected using the Nano-Glo
HiBiT luciferase-based detection system (see ref. 8).
In this section, we detail the delivery of CRISPR/Cas9
reagents by nucleofection (see Note 3) and PCR-based analysis of
the cleavage efficiency of guide RNAs.
3.2.1 Nucleofection
1. Thaw and maintain U2-OS cells using your favorite protocol
(see Note 1). We usually split cells 1:5 every 3–4 days, verify
that the viability is always higher than 95% and limit the number of passages to 20.
2. Two or three days before nucleofection, seed two 75-cm
2 flasks
and incubate at 37
C, 5% CO 2 .
3. When 50–70% confluence is reached, detach the cells with
trypsin, spin-down in a 15-mL tube at 90 g at room temperature for 10 min, remove the supernatant and re-suspend the
cells in PBS to a concentration of 1–3 Â 10
6 cells/mL.
4. Aliquot 10
6 cells into 1.5-mL microcentrifuge tubes (one tube
per guide RNA to be tested plus an additional tube for a
negative control), pellet the cells, discard supernatant
completely, and resuspend cells in Nucleofector
® solution
(100 μL per sample).
5. Prepare the DNA mixes containing 2 μg of guide sgRNA
plasmid (sgXPB) and 2 μg of plasmid expressing the SpCas9
protein (JDS246) in a total volume less than 10 μL.
6. Mix the DNA solution with 100 μL of cell suspension and
transfer to a cuvette. Process the samples quickly to avoid
storing the cells longer than 10 min in Nucleofector
®
Solution V.
7. Insert the cuvette into the Nucleofector
® , select the cell-typespecific program X-001 and press the start button. Using the
provided pipette, immediately remove the sample from the
cuvette and transfer into the six-well plate containing 2 mL of
culture medium.
8. Incubate the six-well plates at 37
C, 5% CO 2 and proceed to
analysis or collect the cells and store the cell pellet at À20
C.
3.2.2 PCR-Based
Analysis of the Cleavage
Efficiency for the sgRNA
CRISPR/Cas9 cleavage is usually assessed by amplifying the target
region by PCR and analyzing the mutation rate in the resulting
product. This can be performed using various assays such as the
polymerase chain reaction (PCR)/restriction enzyme (RE) assay,
T7 endonuclease I (T7EI) assay, Surveyor nuclease assay or with
TIDE [15–18].
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
47