4. Digest pSpCas9(BB)-2A-GFP plasmid with BbsI restriction
enzyme (New England Biolabs Inc, MA) and purified using
QIAquick Gel Extraction Kit (Valencia, CA).
5. The annealed oligos with sgRNA guide sequences were cloned
into the pSpCas9(BB)-2A-GFP plasmid bearing both sgRNA
scaffold backbone (BB) and Cas9 (see Note 3).
6. The plasmid DNA was purified using EndoFree Plasmid Maxi
Kit from Qiagen (Valencia, CA).
2.2 Cell Culture
JEKO-1 cells were obtained from the American Type Culture
Collection (ATCC; www.atcc.org). Cells were maintained in
RPMI-1640 medium supplemented with penicillin, streptomycin,
glutamine, and 10% fetal bovine serum (FBS) and incubated in a
humidified 37
C/5% CO 2 incubator.
2.3 FluorescenceActivated Cell Sorting
(FACS)
S3e™ Cell Sorter (Bio-Rad, Hercules, California) was used for cell
sorting. Alexa 647-conjugated anti-ROR1 antibody was generated
by Thomas Kipps’s lab in the Moores Cancer Center of University
of California, San Diego. Propidium iodide (PI) was purchased
from Thermo Fisher Scientific.
3 Methods
3.1 Electroporation
1. Mix entire supplement with the Nucleofector
® Solution from
Cell Line Nucleofector Kit (VCA-1003, Lonza; Walkersville,
MD).
2. Fill appropriate number of wells with 1.5 ml of supplemented
culture media in 12-well plates and preincubate the plates in a
humidified 37
C/5% CO 2 incubator.
3. Centrifuge 2 Â 10
6 JEKO-1 cells at 200 Â g at room temperature and discard supernatant completely so that no residual
PBS/BSA covers the cell pellet.
4. Mix 100 μl room temperature Nucleofector
® Solution with
2 μg plasmid DNA.
5. Resuspend each cell pellet carefully in 100 μl room temperature
Nucleofector
® Solution and DNA mix (see Note 4).
6. Transfer cell–DNA suspension into the bottom of the cuvette
provided by the kit without air bubbles and close the cuvette
with the cap.
7. Insert the cuvette with cell–DNA suspension into the Nucleofector
® Cuvette Holder and apply the Nucleofector
® Program
X-001.
8. Take the cuvette out of the holder once the program is finished
and add ~500 μl of the preincubated RPMI-1640 media to the
Electroporation of CRISPR-Cas9 into Malignant B Cells for Loss-of-Function. . .
87
enzyme (New England Biolabs Inc, MA) and purified using
QIAquick Gel Extraction Kit (Valencia, CA).
5. The annealed oligos with sgRNA guide sequences were cloned
into the pSpCas9(BB)-2A-GFP plasmid bearing both sgRNA
scaffold backbone (BB) and Cas9 (see Note 3).
6. The plasmid DNA was purified using EndoFree Plasmid Maxi
Kit from Qiagen (Valencia, CA).
2.2 Cell Culture
JEKO-1 cells were obtained from the American Type Culture
Collection (ATCC; www.atcc.org). Cells were maintained in
RPMI-1640 medium supplemented with penicillin, streptomycin,
glutamine, and 10% fetal bovine serum (FBS) and incubated in a
humidified 37
C/5% CO 2 incubator.
2.3 FluorescenceActivated Cell Sorting
(FACS)
S3e™ Cell Sorter (Bio-Rad, Hercules, California) was used for cell
sorting. Alexa 647-conjugated anti-ROR1 antibody was generated
by Thomas Kipps’s lab in the Moores Cancer Center of University
of California, San Diego. Propidium iodide (PI) was purchased
from Thermo Fisher Scientific.
3 Methods
3.1 Electroporation
1. Mix entire supplement with the Nucleofector
® Solution from
Cell Line Nucleofector Kit (VCA-1003, Lonza; Walkersville,
MD).
2. Fill appropriate number of wells with 1.5 ml of supplemented
culture media in 12-well plates and preincubate the plates in a
humidified 37
C/5% CO 2 incubator.
3. Centrifuge 2 Â 10
6 JEKO-1 cells at 200 Â g at room temperature and discard supernatant completely so that no residual
PBS/BSA covers the cell pellet.
4. Mix 100 μl room temperature Nucleofector
® Solution with
2 μg plasmid DNA.
5. Resuspend each cell pellet carefully in 100 μl room temperature
Nucleofector
® Solution and DNA mix (see Note 4).
6. Transfer cell–DNA suspension into the bottom of the cuvette
provided by the kit without air bubbles and close the cuvette
with the cap.
7. Insert the cuvette with cell–DNA suspension into the Nucleofector
® Cuvette Holder and apply the Nucleofector
® Program
X-001.
8. Take the cuvette out of the holder once the program is finished
and add ~500 μl of the preincubated RPMI-1640 media to the
Electroporation of CRISPR-Cas9 into Malignant B Cells for Loss-of-Function. . .
87