cuvette by using the supplied pipettes, and gently transfer the
sample into the preincubated 12-well plate (final volume of
2 ml media per well/sample) (see Note 5).
9. Incubate the cells in humidified 37
C/5% CO 2 incubator until
analysis.
3.2 Screening JEKO1 Cells with ROR1
Knockout
1. Collect the transfected JEKO-1 cells after 48 h and wash the
cells twice with PBS.
2. Resuspend above JEKO-1 cells in 1 ml PBS with 1% FBS.
3. Add 1 μl propidium iodide (PI, 1.0 mg/ml) to JEKO-1 cells
and sort GFP
+ PI
À cells using S3e™ Cell Sorter. The cells are
collected with 2 ml FBS.
4. Centrifuge the cells at 1100 rpm at room temperature, discard
supernatant, and culture the cells in humidified 37
C/5% CO 2
incubator for 2 weeks.
5. Pick up single colonies and culture the cells in humidified
37
C/5% CO 2 incubator until the cell number are enough
for JEKO-1 cells with ROR1 knockout identified by flow cytometry analysis (see Note 6).
4 Notes
1. In our study, the pSpCas9(BB)-2A-GFP plasmid were used and
GFP
+ cells via collected by FACS. Other screening markers,
such as G418 or puromycin, can also be used.
2. Designing the custom sgRNA sequences via the CRISPR
Design Tool is highly recommended (http://tools.genomeengineering.org).
3. Single guide RNA (sgRNA) was cloned into the plasmid
expressing Cas9, which is simple and rapid, involving a single
cloning step with a pair of partially complementary oligonucleotides. sgRNAs can also be delivered as PCR amplicons
containing an expression cassette.
4. Avoid storing the cell suspension longer than 20 min in
Human B Cell Nucleofector
® Solution, as this reduces cell
viability and gene transfer efficiency.
5. Avoid repeated aspiration of the sample, which will reduce the
viability of cells.
6. The transfection efficiency is about 18%, which is enough to
screen GFP
+ PI
À cells (Fig. 1) and subsequent ROR1 knockout
cells (Fig. 2).
88
Jian Yu
sample into the preincubated 12-well plate (final volume of
2 ml media per well/sample) (see Note 5).
9. Incubate the cells in humidified 37
C/5% CO 2 incubator until
analysis.
3.2 Screening JEKO1 Cells with ROR1
Knockout
1. Collect the transfected JEKO-1 cells after 48 h and wash the
cells twice with PBS.
2. Resuspend above JEKO-1 cells in 1 ml PBS with 1% FBS.
3. Add 1 μl propidium iodide (PI, 1.0 mg/ml) to JEKO-1 cells
and sort GFP
+ PI
À cells using S3e™ Cell Sorter. The cells are
collected with 2 ml FBS.
4. Centrifuge the cells at 1100 rpm at room temperature, discard
supernatant, and culture the cells in humidified 37
C/5% CO 2
incubator for 2 weeks.
5. Pick up single colonies and culture the cells in humidified
37
C/5% CO 2 incubator until the cell number are enough
for JEKO-1 cells with ROR1 knockout identified by flow cytometry analysis (see Note 6).
4 Notes
1. In our study, the pSpCas9(BB)-2A-GFP plasmid were used and
GFP
+ cells via collected by FACS. Other screening markers,
such as G418 or puromycin, can also be used.
2. Designing the custom sgRNA sequences via the CRISPR
Design Tool is highly recommended (http://tools.genomeengineering.org).
3. Single guide RNA (sgRNA) was cloned into the plasmid
expressing Cas9, which is simple and rapid, involving a single
cloning step with a pair of partially complementary oligonucleotides. sgRNAs can also be delivered as PCR amplicons
containing an expression cassette.
4. Avoid storing the cell suspension longer than 20 min in
Human B Cell Nucleofector
® Solution, as this reduces cell
viability and gene transfer efficiency.
5. Avoid repeated aspiration of the sample, which will reduce the
viability of cells.
6. The transfection efficiency is about 18%, which is enough to
screen GFP
+ PI
À cells (Fig. 1) and subsequent ROR1 knockout
cells (Fig. 2).
88
Jian Yu