2 weeks. Alternatively, aliquot complete mTeSR™1 medium
and store at À20
C for up to 6 months. Do not freeze-thaw
the complete medium.
3. Always keep Matrigel on ice when thawing and handling to
prevent gelation. The optimal working concentration of Matrigel is cell line dependent. We recommend diluting Matrigel in
ice-cold DMEM/F12 medium at 1:30 to 1:40 dilutions.
Matrigel-coated plates can be used immediately or sealed with
Parafilm to prevent evaporation and stored at 2–8
C for up to a
week. Prior to use, pre-warm the culture plate to room temperature for at least 1 h.
4. Passaging is routinely performed every 4–5 days depending on
the cell line, size of colonies, and plating density. Maintaining
appropriate colony size and density is very important for
keeping a good quality of iPSC culture. Cultures that are too
dense can result in spontaneous differentiation where the colonies lose their border integrity and contain regions of irregular
shape or other cell types. If that occurs, remove the differentiated regions using pipette tip under the inverted microscope.
5. We use the SpCas9 which cuts at 3 nucleotides upstream of the
PAM (5
0 -NGG). Ideally, the cleavage site should be within
10 nucleotides from the mutation to obtain the best targeting
efficiency.
6. The sense and antisense strands contain overhangs (blue color)
for ligation into the pair of BbsI sites in PX459 (CACC and
CAAA, respectively). For transcription from the U6 promoter,
add “G” nucleotide (red color) to the 5
0 end of the sense strand
and its complementary “C” on the antisense strand.
7. Usually, there are no colonies on the negative control plate
(no insert) because the BbsI-digested plasmid alone cannot
reanneal, and there are about hundreds of colonies on the
sgRNA/pX459 cloning plate. Incomplete digestion of pX459
plasmid (uncut plasmid) can also result in colony formation.
Before sequencing the plasmid, quick screening with restriction
enzymes, BbsI and EcoRI, can be performed. The LB plate can
be stored at 4
C (upside down) for up to 1 month.
8. The plasmids should be endotoxin-free and are prepared at a
concentration greater than 2 μg/μL for efficient nucleofection.
The Geneaid™ Midi Plasmid Kit produces endotoxin-free
plasmids. Other kits with similar features can also be used.
9. Gently tap the Nucleocuvette™ strip to make sure that there is
no air bubble in the well before nucleofection. Place the vessel
with closed lid into the retainer of the 4D-Nucleofector™ X
Unit with proper orientation. Do not leave the cells in the
Nucleofection™ solution for extended periods. This will lead
to reduced viability and transfection efficiency.
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
209
and store at À20
C for up to 6 months. Do not freeze-thaw
the complete medium.
3. Always keep Matrigel on ice when thawing and handling to
prevent gelation. The optimal working concentration of Matrigel is cell line dependent. We recommend diluting Matrigel in
ice-cold DMEM/F12 medium at 1:30 to 1:40 dilutions.
Matrigel-coated plates can be used immediately or sealed with
Parafilm to prevent evaporation and stored at 2–8
C for up to a
week. Prior to use, pre-warm the culture plate to room temperature for at least 1 h.
4. Passaging is routinely performed every 4–5 days depending on
the cell line, size of colonies, and plating density. Maintaining
appropriate colony size and density is very important for
keeping a good quality of iPSC culture. Cultures that are too
dense can result in spontaneous differentiation where the colonies lose their border integrity and contain regions of irregular
shape or other cell types. If that occurs, remove the differentiated regions using pipette tip under the inverted microscope.
5. We use the SpCas9 which cuts at 3 nucleotides upstream of the
PAM (5
0 -NGG). Ideally, the cleavage site should be within
10 nucleotides from the mutation to obtain the best targeting
efficiency.
6. The sense and antisense strands contain overhangs (blue color)
for ligation into the pair of BbsI sites in PX459 (CACC and
CAAA, respectively). For transcription from the U6 promoter,
add “G” nucleotide (red color) to the 5
0 end of the sense strand
and its complementary “C” on the antisense strand.
7. Usually, there are no colonies on the negative control plate
(no insert) because the BbsI-digested plasmid alone cannot
reanneal, and there are about hundreds of colonies on the
sgRNA/pX459 cloning plate. Incomplete digestion of pX459
plasmid (uncut plasmid) can also result in colony formation.
Before sequencing the plasmid, quick screening with restriction
enzymes, BbsI and EcoRI, can be performed. The LB plate can
be stored at 4
C (upside down) for up to 1 month.
8. The plasmids should be endotoxin-free and are prepared at a
concentration greater than 2 μg/μL for efficient nucleofection.
The Geneaid™ Midi Plasmid Kit produces endotoxin-free
plasmids. Other kits with similar features can also be used.
9. Gently tap the Nucleocuvette™ strip to make sure that there is
no air bubble in the well before nucleofection. Place the vessel
with closed lid into the retainer of the 4D-Nucleofector™ X
Unit with proper orientation. Do not leave the cells in the
Nucleofection™ solution for extended periods. This will lead
to reduced viability and transfection efficiency.
Correction of HbE/β-Thalassemia iPSCs using CRISPR/Cas9
209