3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. Discard the supernatant and resuspend the pellet with cryopreservation medium at a density of 2 Â 10
6 cells/mL.
5. Quickly dispense 1 mL aliquots of the cell suspension into
each cryogenic vial.
6. Transfer the cryogenic vials into the slow cooling cryocontainer and store at À80
C freezer overnight.
7. The next day, transfer the vials to liquid nitrogen for long-term
storage.
3.3 Preparation
of Plasmids
We generate the GFP knock-in K562 and Raji cells using the
plasmid-based CRISPR/Cas9 system consisting of three individual
plasmids: (1) sgRNA plasmid (gRNA-AAVS1_T2) containing the
sgRNA DNA fragment, which targets AAVS1 locus, under the U6
promoter, (2) Cas9 plasmid (hCas9), and (3) GFP donor plasmid
(AAV-CAGGS-EGFP) harboring the 5
0 and 3
0 homology arms of
AAVS1 gene (~800 bp each), the Puromycin resistant gene, the
CAG promotor, the GFP expression cassette, and the terminator
sequence (poly(A)) (Fig. 1a).
3.3.1 Transformation
and Validation of Plasmids
1. Thaw chemically competent DH5α E. coli cells and plasmids
slowly on ice.
2. Add 100 ng of a plasmid to 50 μL of the chemically competent
DH5α E. coli cells in a 1.5-mL Eppendorf tube.
3. Carefully mix the competent cells with the plasmid using a
pipette and incubate on ice for 10 min.
4. Perform heat shock in a water bath at 42
C for 45 s.
5. Immediately incubate the mixture on ice for 5 min.
6. Add 900 μL of LB medium without ampicillin or kanamycin
and incubate in the shaker at 37
C, at 200 rpm for 1 h.
7. Centrifuge the cell suspension at 500 Â g for 5 min and aspirate
500 μL of supernatant.
8. Gently resuspend the pellet and plate 100 μL of the transformed cells onto the LB agar plates containing 100 μg/mL
ampicillin or 50 μg/mL kanamycin, and incubate the plates
upside down for overnight at 37
C.
9. The next day, pick 5 colonies, inoculate a single colony into
7 mL of LB medium containing 100 μg/mL ampicillin or
50 μg/mL kanamycin, and incubate at 37
C at 200 rpm
overnight.
10. Aliquot 5 mL of culture for plasmid extraction using plasmid
miniprep kit and store 2 mL of culture at 4
C for short-term
storage.
11. Quantify the plasmid yield using NanoDrop 8000.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
221
4. Discard the supernatant and resuspend the pellet with cryopreservation medium at a density of 2 Â 10
6 cells/mL.
5. Quickly dispense 1 mL aliquots of the cell suspension into
each cryogenic vial.
6. Transfer the cryogenic vials into the slow cooling cryocontainer and store at À80
C freezer overnight.
7. The next day, transfer the vials to liquid nitrogen for long-term
storage.
3.3 Preparation
of Plasmids
We generate the GFP knock-in K562 and Raji cells using the
plasmid-based CRISPR/Cas9 system consisting of three individual
plasmids: (1) sgRNA plasmid (gRNA-AAVS1_T2) containing the
sgRNA DNA fragment, which targets AAVS1 locus, under the U6
promoter, (2) Cas9 plasmid (hCas9), and (3) GFP donor plasmid
(AAV-CAGGS-EGFP) harboring the 5
0 and 3
0 homology arms of
AAVS1 gene (~800 bp each), the Puromycin resistant gene, the
CAG promotor, the GFP expression cassette, and the terminator
sequence (poly(A)) (Fig. 1a).
3.3.1 Transformation
and Validation of Plasmids
1. Thaw chemically competent DH5α E. coli cells and plasmids
slowly on ice.
2. Add 100 ng of a plasmid to 50 μL of the chemically competent
DH5α E. coli cells in a 1.5-mL Eppendorf tube.
3. Carefully mix the competent cells with the plasmid using a
pipette and incubate on ice for 10 min.
4. Perform heat shock in a water bath at 42
C for 45 s.
5. Immediately incubate the mixture on ice for 5 min.
6. Add 900 μL of LB medium without ampicillin or kanamycin
and incubate in the shaker at 37
C, at 200 rpm for 1 h.
7. Centrifuge the cell suspension at 500 Â g for 5 min and aspirate
500 μL of supernatant.
8. Gently resuspend the pellet and plate 100 μL of the transformed cells onto the LB agar plates containing 100 μg/mL
ampicillin or 50 μg/mL kanamycin, and incubate the plates
upside down for overnight at 37
C.
9. The next day, pick 5 colonies, inoculate a single colony into
7 mL of LB medium containing 100 μg/mL ampicillin or
50 μg/mL kanamycin, and incubate at 37
C at 200 rpm
overnight.
10. Aliquot 5 mL of culture for plasmid extraction using plasmid
miniprep kit and store 2 mL of culture at 4
C for short-term
storage.
11. Quantify the plasmid yield using NanoDrop 8000.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
221