8. Purify the PCR product using the GenepHlow™ gel/PCR kit
according to the manufacturer’s instruction and further confirm the result with Sanger sequencing.
9. Expand the HbE negative clones and cryopreserve (Subheading 3.1.3) for further analyses.
3.7 Off-Target
Analysis
For off-target analysis, perform PCR amplification of five potential
off-target sites on genomic DNA of the corrected iPSC clones.
1. Extract genomic DNA from the corrected iPSC clones (Subheading 3.6, step 9) using the QuickExtract™ solution.
2. Perform PCR for off-target analysis using the isolated genomic
DNA template, the primers (Table 4), and the PCR reaction
described in Table 9.
3. Transfer the PCR tubes to a thermal cycler using conditions
listed in Table 10.
4. After PCR, run the samples on 1% (w/v) agarose gel at 100 V
for 25 min.
Table 8
Thermal cycling conditions for PCR verification of clones
Step
Temperature and time
Initial denaturation
95
C, 15 min
30 cycles
94
C, 45 s
68
C for 45 s
72
C for 1 min
Final extension
72
C for 7 min
Hold
4
C
1. Wild-type iPSCs or the HbE-corrected iPSCs
2. Patient Eβ-iPSCs
1
2
Internal control (HbE-Fc + Rc, 804 bp)
HbE (HbE-Fm + Rc 529 bp)
Normal control (HbE-Fc + Rn 310 bp)
Fig. 4 Expected results for multiplex PCR analysis of hemoglobin E
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
207