5. 22G needle.
6. 1 mL syringe.
7. Electrospinning setup: Syringe pump, rotating wheel with
speed control, wires, power source.
8. Silicone glue.
9. 70% ethanol.
10. Distilled water (sterilized by 0.02 μm syringe filter).
11. 3,4-Dihydroxy-l-phenylalanine (L-DOPA).
12. Buffer for polymerization of DOPA: 10 mM Bicine, 250 mM
NaCl, HCl (to adjust pH), pH 8.5.
13. Laminin mouse protein.
14. 1Â PBS.
2.2 Preparation
of Complexes
1. sgRNA targeting EGFP: GAAATTAATACGACTCACTA
TAGGTGAACCGCATCGAGCTGAA
GTTTTAGAGCTA
GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
(see Note 1).
2. Cas9 protein: EnGen Cas9 NLS protein (NEB) (see Note 2).
3. Transfection reagent: Lipofectamine CRISPRMAX Cas9
Transfection Reagent (Invitrogen) (see Note 3).
4. Opti-MEM.
2.3 Cell Seeding/
Culture
1. U2OS.EGFP cells.
2. Dulbecco’s Modified Eagle’s Medium (DMEM) with
glutaMAX.
3. Fetal bovine serum (FBS).
4. Penicillin-Streptomycin (10,000 U/mL, PS).
5. Culture medium for U2OS.EGFP cells: DMEM with glutaMAX containing 10% FBS and 1% PS.
6. Trypsin-EDTA (0.05%).
2.4 Evaluation
of EGFP Expression
1. 4% Paraformaldehyde (PFA): 4 g of paraformaldehyde in
100 mL of 1Â PBS.
2. 0.1% Triton-X 100: 0.05 mL of Triton-X 100 in 50 mL of
1Â PBS.
3. Rhodamine Phalloidin.
4. DAPI.
5. 1Â PBS.
6. 0.6 U/mL Proteinase K: working stock in 1Â Tris–EDTA
buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0).
Scaffold-Based Delivery of CRISPR/Cas9
185
6. 1 mL syringe.
7. Electrospinning setup: Syringe pump, rotating wheel with
speed control, wires, power source.
8. Silicone glue.
9. 70% ethanol.
10. Distilled water (sterilized by 0.02 μm syringe filter).
11. 3,4-Dihydroxy-l-phenylalanine (L-DOPA).
12. Buffer for polymerization of DOPA: 10 mM Bicine, 250 mM
NaCl, HCl (to adjust pH), pH 8.5.
13. Laminin mouse protein.
14. 1Â PBS.
2.2 Preparation
of Complexes
1. sgRNA targeting EGFP: GAAATTAATACGACTCACTA
TAGGTGAACCGCATCGAGCTGAA
GTTTTAGAGCTA
GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAT
CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
(see Note 1).
2. Cas9 protein: EnGen Cas9 NLS protein (NEB) (see Note 2).
3. Transfection reagent: Lipofectamine CRISPRMAX Cas9
Transfection Reagent (Invitrogen) (see Note 3).
4. Opti-MEM.
2.3 Cell Seeding/
Culture
1. U2OS.EGFP cells.
2. Dulbecco’s Modified Eagle’s Medium (DMEM) with
glutaMAX.
3. Fetal bovine serum (FBS).
4. Penicillin-Streptomycin (10,000 U/mL, PS).
5. Culture medium for U2OS.EGFP cells: DMEM with glutaMAX containing 10% FBS and 1% PS.
6. Trypsin-EDTA (0.05%).
2.4 Evaluation
of EGFP Expression
1. 4% Paraformaldehyde (PFA): 4 g of paraformaldehyde in
100 mL of 1Â PBS.
2. 0.1% Triton-X 100: 0.05 mL of Triton-X 100 in 50 mL of
1Â PBS.
3. Rhodamine Phalloidin.
4. DAPI.
5. 1Â PBS.
6. 0.6 U/mL Proteinase K: working stock in 1Â Tris–EDTA
buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0).
Scaffold-Based Delivery of CRISPR/Cas9
185