8. Fast digest EcoRI restriction enzyme (Thermo Fisher
Scientific).
9. Gel documentation machine.
10. GenepHlow™ gel extraction and PCR Cleanup kit (Geneaid).
11. 100% glycerol.
12. Ice.
13. Incubating shaker.
14. LB-ampicillin agar: 35 g/L of LB agar, Lennox and 100 μg/
mL ampicillin.
15. LB-ampicillin broth: 25 g/L of Difco™ LB broth and 100 μg/
mL ampicillin.
16. Molecular grade water (ddH 2 O).
17. NanoDrop 2000 (Thermo Fisher Scientific).
18. Novel juice (DNA staining reagent) (GeneDireX).
19. PCR machine T100™ thermal cycler (Bio-Rad).
20. PCR tubes (0.2 mL).
21. pSpCas9(BB)-2A-Puro (PX459) (Addgene) contains the U6
promoter, sgRNA scaffold, SpCas9 and puromycinresistant gene.
22. Sense and antisense strands of sgRNA oligonucleotides with
BbsI overhangs (Table 1).
23. Cell spreader.
24. Standard ladder Lambda DNA/HindIII Marker (Thermo
Fisher Scientific).
25. T4 ligation buffer (Thermo Fisher Scientific).
26. T4 Polynucleotide Kinase (PNK) (Thermo Fisher Scientific) .
27. T7 ligase (New England BioLabs).
28. 10Â Tango buffer (Thermo Fisher Scientific).
29. 1Â TBE buffer: 0.089 M Tris, 0.089 M Borate, 0.002
M EDTA.
Table 1
Oligonucleotide sequences for sgRNA construction
Strand
Oligo sequence (5
0 to 3
0 )
PAM
Sense
CACCGTGGATGAAGTTGGTGGTA
AGG
Antisense
AAACTACCACCAACTTCATCCAC
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
197
Scientific).
9. Gel documentation machine.
10. GenepHlow™ gel extraction and PCR Cleanup kit (Geneaid).
11. 100% glycerol.
12. Ice.
13. Incubating shaker.
14. LB-ampicillin agar: 35 g/L of LB agar, Lennox and 100 μg/
mL ampicillin.
15. LB-ampicillin broth: 25 g/L of Difco™ LB broth and 100 μg/
mL ampicillin.
16. Molecular grade water (ddH 2 O).
17. NanoDrop 2000 (Thermo Fisher Scientific).
18. Novel juice (DNA staining reagent) (GeneDireX).
19. PCR machine T100™ thermal cycler (Bio-Rad).
20. PCR tubes (0.2 mL).
21. pSpCas9(BB)-2A-Puro (PX459) (Addgene) contains the U6
promoter, sgRNA scaffold, SpCas9 and puromycinresistant gene.
22. Sense and antisense strands of sgRNA oligonucleotides with
BbsI overhangs (Table 1).
23. Cell spreader.
24. Standard ladder Lambda DNA/HindIII Marker (Thermo
Fisher Scientific).
25. T4 ligation buffer (Thermo Fisher Scientific).
26. T4 Polynucleotide Kinase (PNK) (Thermo Fisher Scientific) .
27. T7 ligase (New England BioLabs).
28. 10Â Tango buffer (Thermo Fisher Scientific).
29. 1Â TBE buffer: 0.089 M Tris, 0.089 M Borate, 0.002
M EDTA.
Table 1
Oligonucleotide sequences for sgRNA construction
Strand
Oligo sequence (5
0 to 3
0 )
PAM
Sense
CACCGTGGATGAAGTTGGTGGTA
AGG
Antisense
AAACTACCACCAACTTCATCCAC
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
197