silicone glue to dry overnight at room temperature. Prior to
surface modification, store scaffolds in 12-well plates for no
more than a week at room temperature.
9. Sterilize the electrospun fibers by UV irradiation (30 min)
followed by incubation in 70% ethanol for 15 min.
10. Wash scaffolds with sterilized deionized water three times to
rinse away the ethanol.
3.2 Creating
Adhesive Surfaces
with pDOPA
1. To create the pDOPA adhesive surfaces for Cas9:sgRNA
protein-RNA lipofectamine complexes to adhere onto, coat
the electrospun scaffolds with mussel-inspired pDOPA.
2. Dissolve L-DOPA in DOPA polymerization buffer (Subheading 2.1), at a concentration of 0.5 mg/mL.
3. Wait 5 min for L-DOPA to be fully dissolved.
4. Incubate each electrospun scaffold in 1 mL of the DOPA
solution on a shaker (80 rpm) for 2 h at room temperature.
5. After pDOPA coating, wash the electrospun fibers with sterilized deionized water to remove unbound L-DOPA
monomers.
6. Coat laminin onto the scaffolds at 10 μg per scaffold (1 cm
2 )
for 2 h at 37
C. Dilute laminin in sterile 1Â PBS (see Note 10).
3.3 Formation
of Cas9:
sgRNA-Lipofectamine
Complexes
on the Scaffold
1. For Cas9:sgRNA complex formation, mix sgRNA and Cas9
protein in Opti-MEM at a molar ratio of 1:1 according to the
manufacturer’s protocol. Here, lipofectamine CRISPRMAX
is used.
2. For a 1 cm
2 scaffold, dilute 7.5 pmol of sgRNA and Cas9
protein each in 25 μL of Opti-MEM.
3. Thoroughly mix the diluted sgRNA and Cas9 with 2.5 μL of
Cas9 Plus reagent (provided with Lipofectamine
CRISPRMAX).
4. Concurrently, dilute 1.5 μL of Lipofectamine CRISPRMAX
into another microtube with 25 μL of Opti-MEM and incubate
for 1 min at room temperature.
5. Add the mixture with sgRNA, Cas9 protein, and Cas9 Plus
reagent (Cas9-sgRNA complexes) to the diluted Lipofectamine CRISPRMAX and incubate for 15 min at room temperature to form Cas9:sgRNA-lipofectamine complexes.
6. Transfer the mixture onto the scaffold (pDOPA-coated fibers).
Incubate for 1 h at 37
C.
7. Remove the supernatant and wash once with OptiMEM.
8. Seed cells at an appropriate density. For U2OS.EGFP, seed at
10,000 cells per cm
2 in 100 μL. Allow the cells to attach at
37
C before submerging the whole scaffold with an additional
900 μL of culture medium.
Scaffold-Based Delivery of CRISPR/Cas9
187
surface modification, store scaffolds in 12-well plates for no
more than a week at room temperature.
9. Sterilize the electrospun fibers by UV irradiation (30 min)
followed by incubation in 70% ethanol for 15 min.
10. Wash scaffolds with sterilized deionized water three times to
rinse away the ethanol.
3.2 Creating
Adhesive Surfaces
with pDOPA
1. To create the pDOPA adhesive surfaces for Cas9:sgRNA
protein-RNA lipofectamine complexes to adhere onto, coat
the electrospun scaffolds with mussel-inspired pDOPA.
2. Dissolve L-DOPA in DOPA polymerization buffer (Subheading 2.1), at a concentration of 0.5 mg/mL.
3. Wait 5 min for L-DOPA to be fully dissolved.
4. Incubate each electrospun scaffold in 1 mL of the DOPA
solution on a shaker (80 rpm) for 2 h at room temperature.
5. After pDOPA coating, wash the electrospun fibers with sterilized deionized water to remove unbound L-DOPA
monomers.
6. Coat laminin onto the scaffolds at 10 μg per scaffold (1 cm
2 )
for 2 h at 37
C. Dilute laminin in sterile 1Â PBS (see Note 10).
3.3 Formation
of Cas9:
sgRNA-Lipofectamine
Complexes
on the Scaffold
1. For Cas9:sgRNA complex formation, mix sgRNA and Cas9
protein in Opti-MEM at a molar ratio of 1:1 according to the
manufacturer’s protocol. Here, lipofectamine CRISPRMAX
is used.
2. For a 1 cm
2 scaffold, dilute 7.5 pmol of sgRNA and Cas9
protein each in 25 μL of Opti-MEM.
3. Thoroughly mix the diluted sgRNA and Cas9 with 2.5 μL of
Cas9 Plus reagent (provided with Lipofectamine
CRISPRMAX).
4. Concurrently, dilute 1.5 μL of Lipofectamine CRISPRMAX
into another microtube with 25 μL of Opti-MEM and incubate
for 1 min at room temperature.
5. Add the mixture with sgRNA, Cas9 protein, and Cas9 Plus
reagent (Cas9-sgRNA complexes) to the diluted Lipofectamine CRISPRMAX and incubate for 15 min at room temperature to form Cas9:sgRNA-lipofectamine complexes.
6. Transfer the mixture onto the scaffold (pDOPA-coated fibers).
Incubate for 1 h at 37
C.
7. Remove the supernatant and wash once with OptiMEM.
8. Seed cells at an appropriate density. For U2OS.EGFP, seed at
10,000 cells per cm
2 in 100 μL. Allow the cells to attach at
37
C before submerging the whole scaffold with an additional
900 μL of culture medium.
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187