4. QuickExtract™ DNA Extraction Solution (Epicentre).
5. Vortex.
2.6 Off-Target
Analysis
1. Primers for top five off-targets (Table 4).
2. Q5 High-Fidelity DNA polymerase, Q5 PCR buffer with
MgCl 2 and dNTPs (New England Biolabs).
3. QuickExtract™ DNA Extraction Solution (Epicentre).
3 Methods
The following section provides detailed protocols that we use for
the correction of HbE mutation in the Eβ-iPSCs. The protocols
include the culture of iPSCs, design and construction of sgRNA,
cloning of sgRNA into Cas9 expression plasmid, design and preparation of the ssODN repair template, gene targeting of HbE mutation, clonal selection, and verification.
3.1 Culture of iPSCs
iPSCs are maintained under a feeder-free condition in mTeSR™1
medium on Matrigel-coated plates.
3.1.1 Thawing
Cryopreserved iPSCs
1. Warm mTeSR™1 medium to room temperature (see Note 2).
Table 3
Primer sequences for multiplex PCR analysis of hemoglobin E
Primer name
Oligo sequence (5
0
!3
0 )
HbE-Fc
TCCAACTCCTAAGCCAGTGC
HbE-Rn
CCTGCCCAGGGCCTC
HbE-Fm
CGTGGATGAAGTTGGTGGTA
HbE-Rc
CGATCCTGAGACTTCCACACTG
Table 4
Primer sequences for off-target analysis
OT Forward primer (5
0
!3
0 )
Reverse primer (5
0
!3
0 )
Product size (bp)
1
AACCAACCTGCTCACTGGAG
AGCCTTCACCTTAGGGTTGC
309
2
TGGAAGCAGGTGGACAGTTC
TGTGTAGGTTACCCAAGGCAC
369
3
TGCAGGTGTGTAGGTTGAGTC
CCAAACAACCCCTACCACCA
352
4
AACCGCATGGAGTCGTTCTT
ATGGCCCCGAACTAACAGTG
307
5
GAGTTGACCCGAAGAAGCTG
GAGTTGACCCGAAGAAGCTG
412
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
199