3.4 Evaluation
of EGFP Expression
1. After 3 days of culture, fix the cells on scaffolds with 4% PFA for
10 min at room temperature.
2. As cells tend to align and elongate along the aligned electrospun fibers, it may be difficult to detect the outline of cells.
Therefore, additional staining for cellular cytoskeleton may be
needed to count the number of cells for quantifying the
changes in EGFP expression (see Note 11).
3. Add 0.1% Triton-X diluted in PBS to the scaffolds and incubate
for 15 min.
4. Remove the supernatant and stain the cells with Rhodamine
Phalloidin (1:500 in PBS) and DAPI (1:1000 in PBS) for 1 h at
room temperature.
5. Wash the scaffolds three times with PBS.
6. Take images using an epifluorescence or confocal microscope.
7. Successful gene editing will result in knockout of EGFP. The
knockout efficiency of EGFP can be quantified as the percentage of EGFP-positive cells per DAPI. Additional evaluation is
needed to validate the genome editing efficiency (see Note 12).
3.5 Evaluation
of Genomic Cleavage
1. After 3 days of culture, wash the scaffolds once with PBS.
2. Add 200 μL of Proteinase K solution to each scaffold and
incubate the scaffolds in Proteinase K solution for overnight
at 37
C to extract genomic DNA.
3. Transfer the solution from scaffolds to the microtubes (one
scaffold per tube). Flush the fibers with the solution a few times
to ensure genomic DNA is washed out from the scaffolds (see
Note 13).
4. Purify the genomic DNA using a DNA purification kit or
established DNA purification protocol (see Note 4).
5. After the pure genomic DNA is obtained, run a genomic
cleavage assay using a genomic cleavage detection kit or established protocol. During the PCR step to amplify the DNA, use
the Forward and Reverse primers (Subheading 2.4) that are
flanking the EGFP cleavage site (see Notes 5 and 6).
6. Successful genomic cleavage will result in two additional bands
with DNA fragments of smaller sizes (~100 and 300 bp) in
addition to the PCR product of EGFP (~400 bp). Quantify the
cleavage efficiency based on the intensity of the bands on the
gel using the ImageJ “Gel Analyzer” plugin.
4 Notes
1. Successful genome editing depends heavily on a good sgRNA
design. The sequence here has been validated in multiple publications that target EGFP. For other genes, sgRNA has to be
188
Wai Hon Chooi et al.
of EGFP Expression
1. After 3 days of culture, fix the cells on scaffolds with 4% PFA for
10 min at room temperature.
2. As cells tend to align and elongate along the aligned electrospun fibers, it may be difficult to detect the outline of cells.
Therefore, additional staining for cellular cytoskeleton may be
needed to count the number of cells for quantifying the
changes in EGFP expression (see Note 11).
3. Add 0.1% Triton-X diluted in PBS to the scaffolds and incubate
for 15 min.
4. Remove the supernatant and stain the cells with Rhodamine
Phalloidin (1:500 in PBS) and DAPI (1:1000 in PBS) for 1 h at
room temperature.
5. Wash the scaffolds three times with PBS.
6. Take images using an epifluorescence or confocal microscope.
7. Successful gene editing will result in knockout of EGFP. The
knockout efficiency of EGFP can be quantified as the percentage of EGFP-positive cells per DAPI. Additional evaluation is
needed to validate the genome editing efficiency (see Note 12).
3.5 Evaluation
of Genomic Cleavage
1. After 3 days of culture, wash the scaffolds once with PBS.
2. Add 200 μL of Proteinase K solution to each scaffold and
incubate the scaffolds in Proteinase K solution for overnight
at 37
C to extract genomic DNA.
3. Transfer the solution from scaffolds to the microtubes (one
scaffold per tube). Flush the fibers with the solution a few times
to ensure genomic DNA is washed out from the scaffolds (see
Note 13).
4. Purify the genomic DNA using a DNA purification kit or
established DNA purification protocol (see Note 4).
5. After the pure genomic DNA is obtained, run a genomic
cleavage assay using a genomic cleavage detection kit or established protocol. During the PCR step to amplify the DNA, use
the Forward and Reverse primers (Subheading 2.4) that are
flanking the EGFP cleavage site (see Notes 5 and 6).
6. Successful genomic cleavage will result in two additional bands
with DNA fragments of smaller sizes (~100 and 300 bp) in
addition to the PCR product of EGFP (~400 bp). Quantify the
cleavage efficiency based on the intensity of the bands on the
gel using the ImageJ “Gel Analyzer” plugin.
4 Notes
1. Successful genome editing depends heavily on a good sgRNA
design. The sequence here has been validated in multiple publications that target EGFP. For other genes, sgRNA has to be
188
Wai Hon Chooi et al.