Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Summary

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Abstract

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Introduction

Many studies in hematology and immunology rely on the availability of genetically modified mice, including conventional and conditional transgenic/knock-out mice that utilize hematopoietic system-specific Cre drivers such as Mx1-Cre, Vav-Cre, and others^1,2,3,4,5. These strategies require the establishment of new mouse strains, which can be time-consuming and financially burdening. While revolutionary advances in genome editing technology have enabled the generation of new mouse strains in as few as 3-4 mont....

Protocol

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia.

1. Generation and purification of lentivirus particles

NOTE: Lentivirus particles containing the optimized guide RNA can be produced by the detailed protocols provided by Addgene: <https://media.addgene.org/cms/files/Zhang_lab_LentiCRISPR_library_protocol.pdf)>. Optimized methods for high-titer lentivirus preparation and storage are discussed elsewhere^17,18. In brief, lentiviruses are produced by co-tr....

Results

Using the above described protocol, approximately 0.8-1.0 x 10⁸ bone marrow cells per mouse have been obtained. The number of lineage-negative cells we obtain is approximately 3 x 10⁶ cells per mouse. Typically, the yield of bone marrow lineage-negative cells is 4%-5% of that of total bone marrow nuclear cells.

Chimerism of transduced cells (RFP-positive) is evaluated by flow cytometry of the peripheral blo.......

Discussion

The advantage of this protocol is the creation of animal models harboring specific mutations in hematopoietic cells in a rapid and highly cost-effective manner compared to conventional mouse transgenic approaches. It was found that this methodology enables the generation of mice with hematopoietic cell gene-manipulations within 1 month. There are several critical steps in this protocol that require further consideration.

Screening of gRNA sequence

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI Medium 1640 (1X)	Gibco	11875-093	Medium
Sulfamethoxazole and Trimethoprim injection	TEVA	0703-9526-01
1/2 cc LO-DOSE INSULIN SYRINGE	EXELINT	26028	general supply
293T cells	ATCC	CRL-3216--	Cell line
APC-anti-mouse Ly6C (Clone AL-21)	BD Biosciences	560599	Antibodies
APC-Cy7-anti-mouse CD45R (RA3-6B2)	BD Biosciences	552094	Antibodies
BD Luer-Lok disposable syringes, 10 ml	BD	309604	general supply
BD Microtainer blood collection tubes, K2EDTA added	BD Bioscience	365974	general supply
BD Precisionglide needle, 18 G	BD	305195	general supply
BD Precisionglide needle, 22 G	BD	305155	general supply
BV510-anti-mouse CD8a (Clone 53-6.7)	Biolegend	100752	Antibodies
BV711-anti-mouse CD3e (Clone 145-2C11)	Biolegend	100349	Antibodies
Collagen from calf skin	Sigma-Aldrich	9007-34-5	general supply
Corning Costar Ultra-Low Attachment Multiple Well Plate, 6 well	Millipore Sigma	CLS3471	general supply
CRISPR/Cas9 knock-in mice	The Jackson Laboratory	028555	mouse
DietGel 76A	Clear H2O	70-01-5022	general supply
Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucose	Sigma Aldrich	D6429	Medium
eBioscience 1X RBC Lysis Buffer	Thermo fisher Scientific	00-4333-57	Solution
Falcon 100 mm TC-Treated Cell Culture Dish	Life Sciences	353003	general supply
Falcon 5 mL round bottom polystyrene test tube	Life Sciences	352054	general supply
Falcon 50 mL Conical Centrifuge Tubes	Fisher Scientific	352098	general supply
Falcon 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate	Life Science	353046	general supply
Fisherbrand microhematocrit capillary tubes	Thermo Fisher Scientific	22-362566	general supply
Fisherbrand sterile cell strainers, 70 μm	Fisher Scientific	22363548	general supply
FITC-anti-mouse CD4 (Clone RM4-5)	Invitrogen	11-0042-85	Antibodies
Fixation Buffer	BD Bioscience	554655	Solution
Guide-it Compete sgRNA Screening Systems	Clontech	632636	Kit
Isothesia (Isoflurane) solution	Henry Schein	29404	Solution
Lenti-X qRT-PCR Titration Kit	Takara	631235	Kit
Lineage Cell Depletion Kit, mouse	Miltenyi Biotec	130-090-858	Kit
Millex-HV Syringe Filter Unit, 0.45 mm	Millipore Sigma	SLHV004SL	general supply
PBS pH7.4 (1X)	Gibco	10010023	Solution
PE-Cy7-anti-mouse CD115 (Clone AFS98)	eBioscience	25-1152-82	Antibodies
PEI MAX	Polysciences	24765-1	Solution
Penicillin-Streptomycin Mixture	Lonza	17-602F	Solution
PerCP-Cy5.5-anti-mouse Ly6G (Clone 1A8)	BD Biosciences	560602	Antibodies
pLKO5.sgRNA.EFS.tRFP	Addgene	57823	Plasmid
pMG2D	Addgene	12259	Plasmid
Polybrene Infection/Transfection Reagent	Sigma Aldrich	TR-1003-G	Solution
Polypropylene Centrifuge Tubes	BECKMAN COULTER	326823	general supply
psPAX2	Addgene	12260	Plasmid
RadDisk – Rodent Irradiator Disk	Braintree Scientific	IRD-P M	general supply
Recombinant Murine SCF	Peprotech	250-03	Solution
Recombinant Murine TPO	Peprotech	315-14	Solution
StemSpan SFEM	STEMCELL Technologies	09600	Solution
TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coli	Thermo fisher Scientific	K457501	Kit
Zombie Aqua Fixable Viability Kit	BioLegend	423102	Solution