A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Summary

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Abstract

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

Introduction

Precise gene editing forms the cornerstone of major biomedical advances that involve the development of novel strategies to tackle genetic diseases. At the forefront of gene-editing technologies is the method relying on the usage of the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 system that was initially identified as a component of bacterial immunity against the invasion of viral genetic material (reviewed in references^1,2). A major advantage of the CRISPR/Cas9 system over other gene-editing tools, such as zinc-finger nucleases....

Protocol

1. Culturing HEK-293T Cells and Seeding Cells for Transfection

NOTE: Human Embryonic Kidney 293T (HEK-293T) cells are grown in DMEM, high glucose media supplemented with 10% bovine calf serum supplemented with iron and growth promoters, and 1x antibiotic-antimycotic solution (100x solution contains 10,000 units penicillin, 10 mg streptomycin and 25 µg amphotericin B per mL). The media is also supplemented with 1x sodium pyruvate, 1x non-essential amino acid mix, and 2 mM L-Glutamine (stock 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl). Cells are cultured in 100 mm tissue culture plates (approximate growth surface area is 55 cm²). A sub....

Results

Validation of the knockout-efficiency of IDLV-CRISPR/Cas9 vectors
We used GFP-expressing 293T cells as a model to validate the efficiency of CRISPR/Cas9-mediated gene knockout. GFP+ cells were generated by transduction of HEK-293T cells with pLenti-GFP (vBK201a) at an MOI of 0.5 (Figure 3b, "no-virus" panel). The sgRNA-to-GFP/Cas9 all-in-one vector cassette was packaged into IDLV or ICLV particles and the production efficiency was assesse.......

Discussion

IDLVs have begun to emerge as the vehicle of choice for in vivo gene-editing, especially in the context of genetic diseases, owing largely to the low risk of mutagenesis associated with these vectors compared to integrating delivery platforms^22,28. In the current manuscript, we sought to detail the protocol associated with production of the improved all-in-one IDLV-CRISPR/Cas9 system that was recently developed in our laboratory²⁸.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Optima XPN-80 Ultracentrifuge	Beckman Coulter	A99839
Allegra 25R tabletop centrifuge	Beckman Coulter	369434
xMark Microplate Absorbance plate reader	Bio-Rad	1681150
BD FACS	Becton Dickinson	338960
Inverted fluorescence microscope	Leica	DM IRB2
0.45-μm filter unit, 500mL	Corning	430773
Conical bottom ultracentrifugation tubes	Seton Scientific	5067
Conical tube adapters	Seton Scientific	PN 4230
SW32Ti swinging-bucket rotor	Beckman Coulter	369650
15 mL conical centrifuge tubes	Corning	430791
50mL conical centrifuge tubes	Corning	430291
High-binding 96-well plates	Corning	3366
150 mm TC-Treated Cell Culture dishes with 20 mm Grid	Corning	353025
100mm TC-Treated Culture Dish	Corning	430167
0.22 μM filter unit, 1L	Corning	430513
QIAprep Spin Miniprep Kit (50)	Qiagen	27104
Tissue culture pipettes, 5 mL	Corning	4487
Tissue culture pipettes, 10 mL	Corning	4488
Tissue culture pipettes, 25 mL	Corning	4489
Hemacytometer with cover slips	Cole-Parmer	UX-79001-00
Name	Company	Catalog Number	Comments
Cell culture reagents
Human embryonic kidney 293T (HEK 293T) cells	ATCC	CRL-3216
293FT cells	Thermo Fisher Scientific	R70007
DMEM, high glucose media	Gibco	11965
Cosmic Calf Serum	Hyclone	SH30087.04
Antibiotic-antimycotic solution, 100X	Sigma Aldrich	A5955-100ML
Sodium pyruvate	Sigma Aldrich	S8636-100ML
Non-Essential Amino Acid (NEAA)	Hyclone	SH30087.04
RPMI 1640 media	Thermo Fisher Scientific	11875-085
GlutaMAX	Thermo Fisher Scientific	35050061
Trypsin-EDTA 0.05%	Gibco	25300054
BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)	Sigma Aldrich	B9879 - BES
Gelatin	Sigma Aldrich	G1800-100G
Name	Company	Catalog Number	Comments
p24 ELISA reagents
Monoclonal anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	3537
HIV-1 standards	NIH AIDS Research and Reference Reagent Program	SP968F
Polyclonal rabbit anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	SP451T
Goat anti-rabbit horseradish peroxidase IgG	Sigma Aldrich	12-348	Working concentration 1:1500
Normal mouse serum, Sterile, 500mL	Equitech-Bio	SM30-0500
Goat serum, Sterile, 10mL	Sigma	G9023	Working concentration 1:1000
TMB peroxidase substrate	KPL	5120-0076	Working concentration 1:10,000
Name	Company	Catalog Number	Comments
Plasmids
psPAX2	Addgene	12260
pMD2.G	Addgene	12259
pRSV-Rev	Addgene	12253
lentiCRISPR v2	Addgene	52961
Name	Company	Catalog Number	Comments
Restriction enzymes
BsrGI	New England Biolabs	R0575S
BsmBI	New England Biolabs	R0580S
EcoRV	New England Biolabs	R0195S
KpnI	New England Biolabs	R0142S
PacI	New England Biolabs	R0547S
SphI	New England Biolabs	R0182S