Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Summary

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Abstract

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

Introduction

Immune cells are critical for defense against pathogens. Both innate and adaptive immunity are required for clearance of infectants and maintenance of tissue homeostasis^1,2. Cell line models are essential tools for understanding the molecular fundamentals of the mammalian immune system; they are used in in vitro functional assays, such as those modeling human T cell activation, and in determining the function of genetic factors in activating or dampening immune responses^3,4. It is important to note that the mammalian immune system is enormousl....

Protocol

1. Design and Plasmid Construction of sgRNAs Targeting Rosa26 Locus

1. Design guide RNAs around the desired insertion site
   1. Ensure that the insertion site for mouse Rosa26 (hereafter designated as mRosa26) knock-in experiments is located in the first intron of mRosa26; this site has been used in previous studies^28,29. For knock-in experiments in human cells, ensure that the insertion site resides in the human ROSA26 locus (hereafter referred as hROSA26), which has been identified as the human homolog of ....

Results

Following the protocol described above to perform knock-in experiments at the mRosa26 locus using murine RAW264.7 macrophages, we designed a targeting vector to express human ACE2, a receptor for the SARS-Cov-2 virus (Figure 2A). Using a similar design, we generated human Jurkat T cells with knock-in of the OST-tagged RASGRP1 fusion protein (Figure 2C). After transfection of three plasmids, two of which were used for expression of CRISPR/Cas9 (DsRed2; p.......

Discussion

In our experiments, we demonstrated how to perform knock-in editing in immune cells from construct design to knock-in cell screening and validation using human Jurkat T cells and murine RAW264.7 macrophages as examples. Both T cell and macrophage cell lines are resistant to transfection^36,37; however, the problem of low efficiency of CRISPR/Cas9 delivery can be overcome with the aid of fluorescent reporters coupled with cell sorting. This protocol is suitable for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amersham Imager 600	Ge Healthcare		imaging of chemiluminescence
Ampicillin, sodium salt	MP Biomedicals	194526
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	7074	at 1/5000 dilution
Anti-RasGRP1 antibody, clone 10.1	Merck	MABS146	1.0 μg/mL of working concentration
AscI	New England BioLabs	R0558S
β-Actin (D6A8) Rabbit mAb	Cell Signaling Technology	8457	at 1/1000 dilution
BamHI-HF	New England BioLabs	R3136S
BbsI-HF	New England BioLabs	R3539S
Cellometer Mini Automated Cell Counter	Nexcelom Bioscience
E.coli DH5α Competent Cells	Takara	9057
DMSO (Dimethyl Sulfoxide)	MP Biomedicals	196055
DNeasy Blood & Tissue Kits	Qiagen	69506	cell culture reagent
DPBS (10X), no calcium, no magnesium	ThermoFisher Scientific	14200075
Dulbecco's Modified Eagle Medium (DMEM) with high glucose	HyClone	SH30022.01
EcoRI-HF	New England BioLabs	R3101S
FACSAria™ Fusion	BD Biosciences		equipped with biosafety cabinet
FACS Canto flow cytometer	BD Biosciences
Falcon 5 ml polystyrene round bottom test tube	BD Biosciences	352003
Fetal bovine serum (FBS)	ThermoFisher Scientific	10099141
FlowJo version 10.7	BD Biosciences
GAPDH (D16H11) XP Rabbit mAb	Cell Signaling Technology	5174	at 1/1000 dilution
Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP	ThermoFisher Scientific	31430	at 1/5000 dilution
Immobilon ECL Ultra Western HRP Substrate	Millipore	WBKLS0500
Immobilon-PSQ PVDF Membrane	Millipore	ISEQ00010
Jurkat	ATCC	TIB-152	https://www.atcc.org/
Kanamycin sulfate	MP Biomedicals	194531
LB agar powder	ThermoFisher Scientific	22700041
Multi-channel Pipette (30-300 μL)	Eppendorf, or similar
Neon Transfection System	ThermoFisher Scientific	MPK5000
Neon Transfection System, 10 μL kit	ThermoFisher Scientific	MPK1096
Nunc 15 mL Conical Sterile Centrifuge Tubes	ThermoFisher Scientific	339651
OneTaq® Hot Start Quick-Load® 2X Master Mix	New England BioLabs	(M0489)	for high GC% template
PageRuler Prestained Protein Ladder, 10 to 180 kDa	ThermoFisher Scientific	26616
Pipette tip 0.1-20µl	Eppendorf, or similar	0030 075.005
Pipette tip 2-200µl	Eppendorf, or similar	0030 075.021
Pipette tip 50-1000µl	Eppendorf, or similar	0030 075.064
Plasmid Maxi Kit	Qiagen	12163
pX458-DsRed2	Addgene	112219
QIAquick PCR Purification Kit	Qiagen	28104	purify plasmid from restriction digestion
Q5 Hot Start High-Fidelity 2X Master Mix	New England BioLabs	M0494S
RAW264.7	ATCC	TIB-71	https://www.atcc.org/
Recombinant Anti-ACE2 antibody [EPR4435(2)]	Abcam	ab108252	at 1/1000 dilution
RPMI 1640 Medium	HyClone	SH30027.01
Strep-Tactin Sepharose beads	IBA Lifesciences	2-1201-010
Penicillin-Streptomycin	ThermoFisher Scientific	15140122
SYTOX™ Red Dead Cell Stain, for 633 or 635 nm excitation	ThermoFisher Scientific	S34859
T4 DNA ligase	New England BioLabs	M0202S
T4 Polynucleotide Kinase	New England BioLabs	M0201S
Trypan Blue Solution, 0.4%	ThermoFisher Scientific	15250061
Trypsin-EDTA solution (0.25%), with phenol red	ThermoFisher Scientific	25200056
ZOE Fluorescent Cell Imager	Bio-Rad
1.5 mL microtubes, PCR-clean	Eppendorf, or similar	0030 125.215
24-well Clear TC-treated Multiple Well Plates	Corning	3524
96-well Clear Flat Bottom Polystyrene TC-treated Microplates	Corning	3599
96-well Clear Round Bottom TC-treated Microplate	Corning	3799