2 Materials
2.1 Culture of iPSCs
1. We used Eβ-iPSCs for the correction of HbE mutation. The
cell line was derived from skin fibroblasts of β
E
/β
0
-thalassemia
patients [10].
2. Accutase (Merck Millipore).
3. Cell scraper.
4. Cryogenic box.
5. Cryogenic vials (2 mL).
6. Dispase (StemCell Technologies).
7. DMSO.
8. Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12)
(Gibco).
9. Dulbecco’s PBS without Calcium and Magnesium (Gibco).
10. Hemocytometer.
11. Humidified CO 2 incubator.
12. Liquid nitrogen tank.
13. Matrigel
® hESC-qualified Matrix (Corning
® ).
14. Microcentrifuge tubes (0.5 and 1.5 mL).
15. mTeSR™1 medium (StemCell Technologies).
16. 15-mL and 50-mL conical tubes.
17. Sterile seropipettes (5 and 10 mL).
18. Pipette tips (2, 20, 200, and 1000 μL).
19. Tissue culture-treated multi-well plates (6-well, 12-well, and
24-well plates).
20. Rho kinase inhibitor Y-27632 (Calbiochem).
21. 70% ethanol.
2.2 Construction
and Cloning
of Single-Guide
(sgRNA) into Cas9
Expression Plasmid
1. 1% Agarose: 1 g of agarose powder dissolved in 100 mL of 1Â
TBE buffer.
2. 100 μg/mL Ampicillin: working concentration is 100 mg/mL
in ddH 2 O. Store at À20
C.
3. 10 mM ATP (New England BioLabs).
4. Chemically competent DH5α E. coli cells.
5. 10 mM Dithiothreitol (DTT): 1.5 mg of DTT powder dissolved in 1 mL of ddH 2 O.
6. Gel chamber and power source.
7. Fast digest BbsI restriction enzyme (Thermo Fisher Scientific).
196
Methichit Wattanapanitch
2.1 Culture of iPSCs
1. We used Eβ-iPSCs for the correction of HbE mutation. The
cell line was derived from skin fibroblasts of β
E
/β
0
-thalassemia
patients [10].
2. Accutase (Merck Millipore).
3. Cell scraper.
4. Cryogenic box.
5. Cryogenic vials (2 mL).
6. Dispase (StemCell Technologies).
7. DMSO.
8. Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12)
(Gibco).
9. Dulbecco’s PBS without Calcium and Magnesium (Gibco).
10. Hemocytometer.
11. Humidified CO 2 incubator.
12. Liquid nitrogen tank.
13. Matrigel
® hESC-qualified Matrix (Corning
® ).
14. Microcentrifuge tubes (0.5 and 1.5 mL).
15. mTeSR™1 medium (StemCell Technologies).
16. 15-mL and 50-mL conical tubes.
17. Sterile seropipettes (5 and 10 mL).
18. Pipette tips (2, 20, 200, and 1000 μL).
19. Tissue culture-treated multi-well plates (6-well, 12-well, and
24-well plates).
20. Rho kinase inhibitor Y-27632 (Calbiochem).
21. 70% ethanol.
2.2 Construction
and Cloning
of Single-Guide
(sgRNA) into Cas9
Expression Plasmid
1. 1% Agarose: 1 g of agarose powder dissolved in 100 mL of 1Â
TBE buffer.
2. 100 μg/mL Ampicillin: working concentration is 100 mg/mL
in ddH 2 O. Store at À20
C.
3. 10 mM ATP (New England BioLabs).
4. Chemically competent DH5α E. coli cells.
5. 10 mM Dithiothreitol (DTT): 1.5 mg of DTT powder dissolved in 1 mL of ddH 2 O.
6. Gel chamber and power source.
7. Fast digest BbsI restriction enzyme (Thermo Fisher Scientific).
196
Methichit Wattanapanitch