Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

Summary

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

Abstract

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.

Conventional Th17 polarization methods typically involve obtaining naïve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate naïve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

Introduction

Th17 cells, a unique subset of CD4⁺ T helper cells, are vital for eradicating extracellular bacteria and fungi and play a significant role in various autoimmune diseases^1,2,3. Emerging evidence suggests that Th17 cells exhibit heterogeneity, functioning in both pathogenic and non-pathogenic conditions, influenced by environmental and genetic factors. Elucidating the regulatory processes that control the differentiation of Th17 cells, plasticity, and heterogeneity is crucial for the advancement of more effective immunotherapeutic strategies.

Protocol

​All procedures were approved by the Experimental Animal Welfare Ethics Committee, Renji Hospital, Shanghai Jiao Tong University School of Medicine and are in compliance with institutional guidelines.

1. Retroviral production

1.  Preparation of retroviral construct
   1. Use the CRISPick tool (https://portals.broadinstitute.org/gppx/crispick/public) to design the sgRNA for RORγt knockout⁸. Specify the reference gen.......

Discussion

CRISPR/Cas9 genome editing via retroviral delivery is a robust method for exploring the roles of helper T cells. This protocol offers a rapid and effective approach to examining specific genes involved in Th17 differentiation and function. Several critical steps must be carefully followed to achieve optimal results. First, for enhanced gene knockout efficiency, gRNAs should be carefully selected. Given the risk of off-target effects in CRISPR/Cas9 gene editing, it is prudent to choose 2-3 gRNAs with high scores, as ident.......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA (pH 8.0)	Solarbio	E1170
100 mm cell and tissue Culture Dish	BIOFIL	TCD010100
1 M Hepes (Free Acid, sterile)	Solarbio	H1090
24-well cell and tissue culture plate	NEST	702002
48-well cell and tissue culture plate	NEST	748002
Brefeldin A Solution (1,000x)	BioLegend	420601
Brilliant Violet 650 anti-mouse CD4 Antibody (RM4-5)	BioLegend	100546
CD3e Monoclonal Antibody (145-2C11), Functional Grade, eBioscience	Invitrogen	16-0031-82
CD44 Monoclonal Antibody (IM7), PE, eBioscience	Invitrogen	12-0441-83
CD62L (L-Selectin) Monoclonal Antibody (MEL-14), APC, eBioscience	Invitrogen	17-0621-82
Cell counter	Nexcelom Bioscience	Cellometer Auto 2000
Cell Strainer (40 μm)	biosharp	BS-40-CS
Cell Strainer (70 μm)	biosharp	BS-70-CS
Centrifuge	eppendorf	5425 R
Centrifuge	eppendorf	5810 R
ChamQ SYBR Color qPCR Master Mix	Vazyme	Q411-02
ClonExpress II One Step Cloning Kit	Vazyme	C112
DH5α Competent Cells	Sangon Biotech	B528413
Direct-zol RNA Miniprep	ZYMO RESEARCH	R2050
DMEM Medium	BasalMedia	L110KJ
EasySep Mouse CD4+ T Cell Isolation Kit	STEMCELL	19852
eBioscience Fixable Viability Dye eFluor 660	Invitrogen	65-0864-18
EndoFree Mini Plasmid Kit II	TIANGEN	DP118-02
ExFect Transfection Reagent	Vazyme	T101-01
Fetal Bovine Serum, Premium Plus	Gibco	A5669701
FITC anti-mouse IL-17A Antibody (TC11-18H10.1)	BioLegend	506907
Formaldehyde solution	Macklin	F864792
HiScript IV RT SuperMix for qPCR（+gDNA wiper）	Vazyme	R423-01
Ionomycin	Beyotime	S1672
Mitomycin C	Maokang Biotechnology	7/7/1950
Mouse GRCm38	NCBI	RefSeq v.108.20200622
OPTI-MEM Reduced Serum Medium	Gibco	31985070	reduced serum medium
Pacific Blue anti-mouse CD4 Antibody (RM4-5)	BioLegend	100531
PE/Cyanine7 anti-mouse CD25 Antibody (PC61)	BioLegend	102015
PE/Cyanine7 anti-mouse CD4 Antibody (GK1.5)	BioLegend	100422
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
PMA/TPA	Beyotime	S1819
R26-CAG-Cas9 mice	Shanghai Model Organisms Center	Cat. NO. NM-KI-00120
Recombinant Human TGF-beta 1 (CHO-Expressed) Protein, CF	R&D Systems	11409-BH
Recombinant Murine IL-6	PeproTech	216-16
Research Cell Analyzer	BD Biosciences	BD LSRFortessa
Research Cell Sorter	SONY	MA900
RPMI 1640 Medium	BasalMedia	L210KJ
SimpliAmp Thermal Cycler PCR System	Applied Biosystems	A24811
Sodium pyruvate solution (100 mM)	Sigma-Aldrich	S8636
Ultra-LEAF Purified anti-mouse CD28 Antibody (37.51)	BioLegend	102121
Ultra-LEAF Purified anti-mouse IFN-γ Antibody (XMG1.2)	BioLegend	505847
Ultra-LEAF Purified anti-mouse IL-4 Antibody (11B11)	BioLegend	504135
Ultra-LEAF Purified anti-mouse IL-12/IL-23 p40 Antibody (C17.8)	BioLegend	505309
β-Mercaptoethanol (50 mM)	Solarbio	M8211