to repair the DSB. This mechanism can be exploited to generate
precise gene modifications [16, 18].
In this chapter, we describe the protocol for precise GFP gene
knock-in in K562 and Raji cells at a genomic safe harbor locus
(AAVS1) using a plasmid-based CRISPR/Cas9 technology. Upon
transfection with the CRISPR/Cas9 and the GFP donor plasmids,
both of the transfected cells express high levels of GFP. Further
enrichment using fluorescence-activated cell sorting (FACS) or
limiting dilution generates a homogeneous population of GFP
+
cells, which can be directly used for NK cell killing assay. Our
approach is easily performed and applicable to the GFP knock-in
in various target cell types for functional analysis of effector cells.
2 Materials
2.1 Culture of K562,
Raji, and NK-92
Cell Line
1. K562 and Raji cell culture medium: RPMI-1640, 10% FBS,
2 mM GlutaMAX™, 100 U/mL Penicillin-Streptomycin.
2. K562 and Raji cell cryopreservation medium: 90% FBS and
10% DMSO.
3. NK-92 cell culture medium: α-MEM, 10% FBS, 10% horse
serum, 2 mM GlutaMAX™, 100 U/mL PenicillinStreptomycin, 100 U/mL hIL-2.
4. NK-92 cell cryopreservation medium: 50% FBS, 40% NK-92
cell culture medium, and 10% DMSO.
5. 70% ethanol.
6. 15-mL conical tubes.
7. 25-cm
2 cell culture flasks.
8. Slow cooling cryo-container.
9. Cryogenic vials.
10. Hemocytometer.
11. Sterile seropipettes, pipette tips (200 and 1000 μL).
12. Tissue culture hood and humidified incubator (37
C, 5%
CO 2 ).
13. Inverted microscope.
14. Liquid nitrogen tank.
2.2 Preparation
of Plasmids
1. Chemically competent DH5α E. coli cells.
2. gRNA_AAVS1-T2 (Addgene): sgRNA plasmid.
3. Plasmid hCas9 (Addgene): Cas9 plasmid.
4. AAV-CAG-EGFP (Addgene): GFP donor plasmid.
5. Glycerol stock of DH5α E. coli transformed with sgRNA plasmid, Cas9 plasmid, and GFP donor plasmid.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
215
precise gene modifications [16, 18].
In this chapter, we describe the protocol for precise GFP gene
knock-in in K562 and Raji cells at a genomic safe harbor locus
(AAVS1) using a plasmid-based CRISPR/Cas9 technology. Upon
transfection with the CRISPR/Cas9 and the GFP donor plasmids,
both of the transfected cells express high levels of GFP. Further
enrichment using fluorescence-activated cell sorting (FACS) or
limiting dilution generates a homogeneous population of GFP
+
cells, which can be directly used for NK cell killing assay. Our
approach is easily performed and applicable to the GFP knock-in
in various target cell types for functional analysis of effector cells.
2 Materials
2.1 Culture of K562,
Raji, and NK-92
Cell Line
1. K562 and Raji cell culture medium: RPMI-1640, 10% FBS,
2 mM GlutaMAX™, 100 U/mL Penicillin-Streptomycin.
2. K562 and Raji cell cryopreservation medium: 90% FBS and
10% DMSO.
3. NK-92 cell culture medium: α-MEM, 10% FBS, 10% horse
serum, 2 mM GlutaMAX™, 100 U/mL PenicillinStreptomycin, 100 U/mL hIL-2.
4. NK-92 cell cryopreservation medium: 50% FBS, 40% NK-92
cell culture medium, and 10% DMSO.
5. 70% ethanol.
6. 15-mL conical tubes.
7. 25-cm
2 cell culture flasks.
8. Slow cooling cryo-container.
9. Cryogenic vials.
10. Hemocytometer.
11. Sterile seropipettes, pipette tips (200 and 1000 μL).
12. Tissue culture hood and humidified incubator (37
C, 5%
CO 2 ).
13. Inverted microscope.
14. Liquid nitrogen tank.
2.2 Preparation
of Plasmids
1. Chemically competent DH5α E. coli cells.
2. gRNA_AAVS1-T2 (Addgene): sgRNA plasmid.
3. Plasmid hCas9 (Addgene): Cas9 plasmid.
4. AAV-CAG-EGFP (Addgene): GFP donor plasmid.
5. Glycerol stock of DH5α E. coli transformed with sgRNA plasmid, Cas9 plasmid, and GFP donor plasmid.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
215