Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

Abstract

Source: Sterner, R. M. et al., Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells. J. Vis. Exp. (2019)

The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.

Protocol

1. CART19 cell production

1. T-cell isolation, stimulation, and ex-vivo culture
   1. Carry out all cell culture work in a cell culture hood utilizing appropriate personal protective equipment. Harvest peripheral blood mononuclear cells (PBMCs) from de-identified normal donor blood cones collected during apheresis as these are known to be a viable source of PBMCs.
   2. To isolate PBMCs, add 15 mL of a density gradient medium to a 50 mL density gradient separation tube. Dilute the donor blood with an equal volume of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) to avoid cell trapping.
      1. Add the ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CD3 Monoclonal Antibody (OKT3), PE, eBioscience	Invitrogen	12-0037-42
CD3 Monoclonal Antibody (UCHT1), APC, eBioscience	Invitrogen	17-0038-42
Choice Taq Blue Mastermix	Denville Scientific	C775Y51
CTS (Cell Therapy Systems) Dynabeads CD3/CD28	Gibco	40203D
CytoFLEX System B4-R2-V2	Beckman Coulter	C10343	Flow cytometer
Dimethyl sulfoxide	Millipore Sigma	D2650-100ML
Dulbecco's Phosphate-Buffered Saline	Gibco	14190-144
Dynabeads MPC-S (Magnetic Particle Concentrator)	Applied Biosystems	A13346
Easy 50 EasySep Magnet	STEMCELL Technologies	18002
EasySep Human T Cell Isolation Kit	STEMCELL Technologies	17951	Negative selection magnetic beads; 17951RF includes tips and buffer
Fetal bovine serum	Millipore Sigma	F8067
FITC Mouse Anti-Human CD107a	BD Pharmingen	555800
Fixation Medium (Medium A)	Invitrogen	GAS001S100
GenCRISPR gRNA Construct: Name: CSF2	GenScript	N/A	Custom order
CRISPR guide RNA 1; Species: Human, Vector:
pLentiCRISPR v2; Resistance: Ampicillin; Copy number:
High; Plasmid preparation: Standard delivery: 4 μg (Free
of charge)
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Invitrogen	A-21235
https://tide.nki.nl.	Desktop Genetics
Human AB Serum; Male Donors; type AB; US	Corning	35-060-CI
IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience	Invitrogen	47-7319-42
Lipofectamine 3000 Transfection Reagent	Invitrogen	L3000075
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation	Invitrogen	L34966
Lymphoprep	STEMCELL Technologies	7851
Monensin Solution, 1000X	BioLegend	420701
Mouse Anti-Human CD28 Clone CD28.2	BD Pharmingen	559770
Mouse Anti-Human CD49d Clone 9F10	BD Pharmingen	561892
Mouse Anti-Human MIP-1β PE-Cy7	BD Pharmingen	560687
Mr. Frosty Freezing Container	Thermo Scientific	5100-0001
NALM6, clone G5	ATCC	CRL-3273	Acute lymphoblastic leukemia cell line
Nuclease Free Water	Promega	P119C
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile	Genesee Scientific	25-227
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile	Genesee Scientific	25-228
Opti-MEM I Reduced-Serum Medium (1X), Liquid	Gibco	31985-070
PE-CF594 Mouse Anti-Human IL-2	BD Horizon	562384
Penicillin-Streptomycin-Glutamine (100X), Liquid	Gibco	10378-016
Permeabilization Medium (Medium B)	Invitrogen	GAS002S100
PureLink Genomic DNA Mini Kit	Invitrogen	K182001
Puromycin Dihydrochloride	MP Biomedicals, Inc.	210055210
QIAquick Gel Extraction Kit	QIAGEN	28704
Rat Anti-Human GM-CSF BV421	BD Horizon	562930
RoboSep-S	STEMCELL Technologies	21000	Fully Automated Cell Separator
SepMate-50 (IVD)	STEMCELL Technologies	85450
Sodium Azide, 5% (w/v)	Ricca Chemical	7144.8-16
X-VIVO 15 Serum-free Hematopoietic Cell Medium	Lonza	04-418Q