Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation

Summary

We outline a highly adaptable approach to using CRISPR-Cas9 ribonucleoprotein complex-mediated gene ablation in murine naïve CD4 T cells to investigate gene function in CD4 T cell differentiation.

Abstract

The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies.

Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals.

Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.

Introduction

The use of clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies has transformed our ability to manipulate genomic DNA sequences, greatly enhancing our ability to study gene function in countless biological systems. With respect to CD4 T cells, methods utilizing CRISPR-Cas9 ribonucleoprotein (RNP) complexes have emerged, facilitating efficient gene knock-out (KO) in primary naïve T cells that can be used for in vitro and in vivo studies^1,2,3,4. Identification of new putative genes regulat....

Protocol

For all procedures described here, we used wild-type (WT) C57/BL6J mice. Mice were maintained and treated under specific pathogen-free (SPF) conditions in accordance with the guidelines of NIAID (protocol LISB-22E) and the Animal Care and Use committees at the NIH (Animal Welfare Assurance #A-4149-01).

1. Considerations before beginning

1. Selecting gRNA reagents
   NOTE: See the Table of Materials for obtaining predesigned gRNA reagents targeting the mouse genome used in this protocol.
   1. Search the gene of interest (Table of Materials, Desired crRNAs) and select the ....

Results

To validate that a pure population of naïve CD4 T cells was obtained using our protocol (Section 2), we used flow cytometry to identify these cells before and after magnetic isolation. Using our approach, we obtained a highly pure population of live CD4⁺TCRb⁺CD25^-CD44^-CD62L⁺ naïve CD4 T cells following isolation (Figure 3A). Furthermore, to confirm that Cas9-gRNA complexes were successfully nucleofected into naïve CD4 T .......

Discussion

Integrating protocols for delivering CRISPR-Cas9 complexes into naïve CD4 T cells with methods for studying CD4 T cell differentiation provides a robust tool to explore novel genes that regulate CD4 T cell biology. Here, we provide a comprehensive guide for utilizing commercially available Cas9 and gRNA reagents that are straightforward to work with. Nucleofection-mediated delivery of Cas9-gRNA complexes into naïve CD4 T cells provides highly efficient gene editing, facilitating near-total knockout of genes of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH = 8.0	IPM Scientific	11005-016
16% Paraformaldehyde, PFA	Electron Microscopy Sciences	15710	Dilute with PBS to make 4% PFA fixative solution
1x eBioscience Buffer (1x Permeabilization buffer)	Thermo Fisher Scientific	00-5523-00	Use permeabilization buffer from kit to make 1x solution
1x PBS, pH = 7.4	Quality Biological	114-058-101
2-mercaptoethanol (1000x)	Gibco	21985-023
4D Nucleofector Core Unit	Lonza	AAF-1003B
4D Nucleofector X Unit	Lonza	AAF-1003X
60 mm dish	Falcon	353002
70 µm nylon mesh strainer	Fisherbrand	22363548
ACK Lysing Buffer	Gibco	A10492-01
Amaxa P3 Primary Cell 4D-Nucleofector X Kit	Lonza	V4XP-3032	This kit includes: P3 Primary Cell Solution, Supplement 1, and 16-well nucleocuvette strips
anti-FITC microbeads	Miltenyi Biotec	130-048-701	Follow Miltenyi Anti-FITC Microbeads Protocol
anti-mouse CD28 (37.51)	bioXcell	BE0015-1	see Table 1 for stock concentration
anti-mouse CD3e (145-2C11)	bioXcell	BE0001-1	see Table 1 for stock concentration
anti-mouse IFNγ (XMG1.2)	bioXcell	BE0055	see Table 1 for stock concentration
anti-mouse IL-12p40 (C17.8)	bioXcell	BE0051	see Table 1 for stock concentration
anti-mouse IL-4 (11B11)	bioXcell	BE0045	see Table 1 for stock concentration
BioLite 48-well Mutlidish	Thermo Fisher Scientific	130187
Bovine Serum Albumin, BSA	Sigma Life Science	A3059
Cas9 enzyme	IDT	1081059	Alt-R S.p. Cas9 Nuclease V3 (10mg/mL) - contains nuclear localization sequence
Complete IMDM + IL-7 Media			Complete IMDM with 5 ng/mL IL-7
Complete IMDM Media			IMDM+GlutMAX, 10% FBS, 1% L-Glutamine, 1% Pen/Strep, 0.1% BME
Complete RPMI Media			RPMI-1640, 10% FBS, 1% L-Glutamine, 1% Pen/Strep, 0.1% BME
CRISPick			https://portals.broadinstitute.org/gppx/crispick/public
Desired crRNAs	IDT		Used predesigned from IDT website: https://www.idtdna.com/site/order/designtool/index/CRISPR_PREDESIGN
eBioscience FOXP3/Transcription Factor Staining Buffer Set	Thermo Fisher Scientific	00-5523-00	The kit contains three reagents: Fixation/Permeabilization Concentrate (4x), Fixation/Permeabilization Diluent, and Permeabilization Buffer (10x)
FACS Buffer			1x PBS, 1% FBS, 1 mM EDTA
Fetal Bovine Serum (FBS)	VWR Seradigm Life Science	97068-085	Heat inactivate prior to use (warm to 56°C for 45 minutes)
FITC anti-mouse CD4 (RM4-5)	Biolegend	100510	1/200 dilution (0.5 mg/mL stock concentration)
FITC anti-mouse CD8α (53-6.7)	Biolegend	100706	1/200 dilution (0.5 mg/mL stock concentration)
Golgi Stop	BD Biosciences	51-2092KZ	1/2000 dilution
IMDM (1x) + GlutMAX-1 Media	Gibco	31980-030
Ionomycin calcium salt from Streptomyces conglobatus (1 mg/mL)	Sigma Aldrich	10634	Recommended final concentration of 500 ng/mL
L-Glutamine 200 mM (100x)	Gibco	25030-081
LS columns	Miltenyi Biotec	130-042-401
MACS Buffer			1x PBS, 0.5% BSA, 1 mM EDTA, filter sterilized
Mitomycin C (0.5 mg/mL)	Millipore Sigma	M4287-2MG
Mouse Naïve CD4 T Cell Isolation Kit	Miltenyi Biotec	130-104-453	Follow Miltenyi Naïve CD4 T Cell Isolation Protocol. This kit includes Naïve CD4+ T Cell Biotin Antibody Cocktail, Anti-Biotin Microbeads and CD44 Microbeads
Negative control crRNA	IDT	1072544	alternative to designing own negative control
Nuclease Free Duplex Buffer	IDT	1072570
PCR tube strips	USA Scientific	1402-2700
Penicillin Streptomycin	Gibco	15140-023
Phorbol 12-myristate 13-acetate, PMA (100 µg/mL)	Sigma Aldrich	P8139	Recommended final concentration of 50 ng/mL
ProSeries High Performance 15mL Centrifuge Tubes	Alkali Scientific	PS5600	15 mL conical tubes
recombinant human (h) IL-2	Peprotech	200-02	see Table 1 for stock concentration
recombinant human TGF-b1 (HEK293 derived)	Peprotech	100-21	see Table 1 for stock concentration
recombinant murine IL-12p70	Peprotech	210-12	see Table 1 for stock concentration
recombinant murine IL-4	Peprotech	214-14	see Table 1 for stock concentration
recombinant murine IL-6	Peprotech	216-16	see Table 1 for stock concentration
recombinant murine IL-7	Peprotech	217-17	Prepare at 100 ng/mL
RPMI 1640 Media	Gibco	21870-076
Thermal Cycler	Applied Biosystems	4375786	We use this model of thermocycler, however any similar equipment will work well in this protocol
tracrRNA Atto550 labeled	IDT	1075928	Allows detection of Cas9-gRNA complexes after nuceloefection using Atto550 fluoresence as a readout. We recommend this reagent if feasible.
Triton-X Buffer			1x PBS, 0.5% TritonX-100, 0.1% BSA
TritonX-100	BioRad	161-0407
unlabeled tracrRNA	IDT	1072534	A more cost effective tracrRNA option, but does not permit evaluation of nucelofection efficiency of Cas9-gRNA complexes
Veriti Thermal Cycler, 96-well Fast	Thermo Fisher Scientific	4375305	We use this model of thermocycler, however any similar equipment will work well in this protocol