12. Incubate the transfected cells at 37
C, 5% CO 2 .
13. Refeed the cells daily with mTeSR™1 medium after 24 h postnucleofection.
3.5 Clonal Isolation
of Genetic Corrected
iPSCs
Clonal isolation of the transfected iPSCs can be performed as early
as 24 h post-transfection by FACS or serial dilutions. This section
provides the protocol for clonal isolation using serial dilutions. We
obtain better viability using serial dilutions as compared to FACS.
1. At day 3 post-transfection, pre-treat the transfected cells with
SMC4 in mTeSR™1 medium for an hour (see Note 10).
2. Prepare Matrigel-coated 96-well plates for seeding (see Note
3).
3. Remove the mTeSR™1 medium from the 24-well cultured
plate (step 1) and wash the cells once with DPBS.
4. Dissociate the cells using 0.25 mL of Accutase at 37
C for 7
min.
5. Add 0.25 mL of mTeSR™1 medium supplemented
with SMC4.
6. Centrifuge the cells at 500 Â g for 5 min at room temperature.
Filter the cells using 40-μm cell strainer.
7. Resuspend the cells in mTeSR™1 medium supplemented with
SMC4. Dilute the cells and seed the cells at a density of 10 cells
in 100 μL/well of 96-well plates (see Note 11).
8. Seed at least two 96-well plates and incubate at 37
C, 5% CO 2 .
9. The next day, examine the plate under a microscope. Identify
the well that contains a single cell and mark off the wells that
are empty or contain multiple cells.
10. Culture the cells in mTeSR™1 medium supplemented with
SMC4 for 8 days.
11. Expand the cells for 2 weeks or until the size of the colony is
large enough for picking.
12. Manually pick half of the single colony using a 200-μL pipette
tip for genomic DNA isolation and genotyping (see Subheading 3.6).
13. To establish a back-up culture, pick up the remaining half of
the colony and transfer to a well of a Matrigel-coated 12-well
plate (see Note 3) containing the pre-warmed mTeSR™1
medium supplemented with 10 μM Y-27632.
3.6 Verification
of Clones by Multiplex
PCR Analysis
of Hemoglobin E
The following protocol describes isolation of genomic DNA from
the iPSC clones for genotyping after gene editing.
1. Transfer half of the colony from Subheading 3.5 (step 12) to a
0.5-mL tube containing 10 μL of QuickExtract™ solution.
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
205
C, 5% CO 2 .
13. Refeed the cells daily with mTeSR™1 medium after 24 h postnucleofection.
3.5 Clonal Isolation
of Genetic Corrected
iPSCs
Clonal isolation of the transfected iPSCs can be performed as early
as 24 h post-transfection by FACS or serial dilutions. This section
provides the protocol for clonal isolation using serial dilutions. We
obtain better viability using serial dilutions as compared to FACS.
1. At day 3 post-transfection, pre-treat the transfected cells with
SMC4 in mTeSR™1 medium for an hour (see Note 10).
2. Prepare Matrigel-coated 96-well plates for seeding (see Note
3).
3. Remove the mTeSR™1 medium from the 24-well cultured
plate (step 1) and wash the cells once with DPBS.
4. Dissociate the cells using 0.25 mL of Accutase at 37
C for 7
min.
5. Add 0.25 mL of mTeSR™1 medium supplemented
with SMC4.
6. Centrifuge the cells at 500 Â g for 5 min at room temperature.
Filter the cells using 40-μm cell strainer.
7. Resuspend the cells in mTeSR™1 medium supplemented with
SMC4. Dilute the cells and seed the cells at a density of 10 cells
in 100 μL/well of 96-well plates (see Note 11).
8. Seed at least two 96-well plates and incubate at 37
C, 5% CO 2 .
9. The next day, examine the plate under a microscope. Identify
the well that contains a single cell and mark off the wells that
are empty or contain multiple cells.
10. Culture the cells in mTeSR™1 medium supplemented with
SMC4 for 8 days.
11. Expand the cells for 2 weeks or until the size of the colony is
large enough for picking.
12. Manually pick half of the single colony using a 200-μL pipette
tip for genomic DNA isolation and genotyping (see Subheading 3.6).
13. To establish a back-up culture, pick up the remaining half of
the colony and transfer to a well of a Matrigel-coated 12-well
plate (see Note 3) containing the pre-warmed mTeSR™1
medium supplemented with 10 μM Y-27632.
3.6 Verification
of Clones by Multiplex
PCR Analysis
of Hemoglobin E
The following protocol describes isolation of genomic DNA from
the iPSC clones for genotyping after gene editing.
1. Transfer half of the colony from Subheading 3.5 (step 12) to a
0.5-mL tube containing 10 μL of QuickExtract™ solution.
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
205