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 months with the appropriate technical expertise^6,7,8,9, much more time is required to amplify the mouse colony before experiments are pursued. In addition, these procedures are costly. For example, Jackson Laboratory lists the current price of knock-out mice generation services at $16,845 per strain (as of December 2018). Thus, methods that are more economical and efficient than conventional murine transgenic approaches are more advantageous.

Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology has led to the development of new tools for rapid and efficient RNA-based, sequence-specific genome editing. Originally discovered as a bacterial adaptive immune mechanism to destroy invading pathogen DNA, the CRISPR/Cas9 system has been used as a tool to increase the effectiveness of genome editing in eukaryotic cells and animal models. A number of approaches have been employed to transmit CRISPR/Cas9 machinery into hematopoietic stem cells (i.e., electroporation, nucleofection, lipofection, viral delivery, and others).

Here, a lentivirus system is employed to transduce cells due to its ability to effectively infect Cas9-expressing murine hematopoietic stem cells and package together the guide RNA expression construct, promoters, regulatory sequences, and genes that encode fluorescent reporter proteins (i.e., GFP, RFP). Using this method, ex vivo gene editing of mouse hematopoietic stem cells has been achieved, followed by successful reconstitution of bone marrow in lethally irradiated mice¹⁰. The lentivirus vector employed for this study expresses the Cas9 and GFP reporter genes from the common core EF1a promoter with an internal ribosomal entry site upstream from the reporter gene. The guide RNA sequence is expressed from a separate U6 promoter. This system is then used to create insertion and deletion mutations in the candidate clonal hematopoiesis driver genes Tet2 and Dnmt3a¹⁰. However, the transduction efficiency by this method is relatively low (~5%-10%) due to the large size of the vector insert (13 Kbp) that limits transduction efficiency and reduces virus titer during production.

In other studies, it has been shown that larger viral RNA size negatively affects both virus production and transduction efficiency. For example, a 1 kb increase in insert size is reported to decrease virus production by ~50%, and transduction efficiency will decrease to more than 50% in mouse hematopoietic stem cells¹¹. Thus, it is advantageous to reduce the size of the viral insert as much as possible to improve efficiency of the system.

This shortcoming can be overcome by employing Cas9 transgenic mice, in which the Cas9 protein is expressed in either a constitutive or inducible manner¹². The constitutive CRISPR/Cas9 knock-in mice expresses Cas9 endonuclease and EGFP from the CAG promoter at the Rosa26 locus in a ubiquitous manner. Thus, a construct with sgRNA under the control of the U6 promoter and RFP reporter gene under the control of the core EF1a promoter can be delivered using the lentivirus vector to achieve genome editing. With this system, the genes of hematopoietic stem cells have been successfully edited, showing a ~90% transduction efficiency. Thus, this protocol provides a rapid and effective method to create mice in which targeted gene mutations are introduced into the hematopoietic system. While our lab is predominantly using this type of technology to study the role of clonal hematopoiesis in cardiovascular disease processes^13,14,15, it is also applicable to studies of hematological malignancy¹⁶. Furthermore, this protocol can be extended to the analysis of how DNA mutations in HSPC impact other disease or developmental processes in the hematopoietic system.

To establish a robust lentivirus vector system, high titer viral stocks and optimized conditions for the transduction and transplantation of hematopoietic cells are required. In the protocol, instructions are provided on the preparation of a high titer viral stock in section 1, optimizing the culture conditions of murine hematopoietic stem cells in section 2, methods for bone marrow transplantation in section 3, and assessing engraftment in section 4.

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-transfection of a lentivirus vector plasmid, psPAX2, and pMD2.G into HEK 293T cells. Culture supernatant is collected at 48 h post-transfection and concentrated by ultracentrifugation. Lentiviral titer is determined by a commercially available qPCR-based assay. This procedure should be performed in a biosafety class II cabinet.

1. Prepare a 1:200 solution of collagen (0.0005%) in 1x PBS.
2. Coat a 6 well plate with collagen solution and incubate at 37 °C, 5% CO₂ for ~30 min.
3. Seed 293T cells at a density of 1 x 10⁶ cells per well and incubate at 37 °C, 5% CO₂ for ~2 h.
4. To prepare the mixture of three transfection plasmids for one well, combine 0.9 µg of lentivirus vector, 0.6 µg of psPAX2, and 0.3 µg of pMD2.G, then achieve a total volume of 10 µL by adding deionized water. Adjust amounts accordingly depending on the number of wells. The amount and ratio of each plasmid may need to be further optimized to suit the researchers needs.
5. Carefully add 50 µL of 1x PBS and 5 µL of the diluted PEI MAX (1.0 mg/mL) to the plasmid mixture and incubate for 15 min at room temperature (RT) (Table 1).
6. Add 1 mL of DMEM to the mixture.
7. Aspirate media from the 6 well plate, add 1 mL of plasmid mixture, and incubate at 37 °C, 5% CO₂ for ~3 h.
8. Replace the media with 2 mL of fresh DMEM and incubate at 37 °C, 5% CO₂ for 24 h.
9. Add 1 mL of fresh DMEM and incubate at 37 °C, 5% CO₂ for an additional 24 h (total incubation time is 48 h).
10. Transfer the culture supernatant to a 50 mL tube and centrifuge at 3,000 x g for 15 min to remove any free-floating cells.
11. Filter the supernatant through a 0.45 µm filter.
12. Transfer the filtrate to polypropylene centrifuge tubes.
13. Ultracentrifuge at 4 °C and 72,100 x g at r_max for 3 h.
14. Carefully aspirate the supernatant, leaving behind the white pellet.
15. Resuspend the pellet with 100 µL of serum-free hematopoietic cell expansion medium without aeration.
16. Keep a 10 µL aliquot to measure the viral titer and store all remaining aliquots at -80 °C until required.
17. Titrate the virus with a qPCR-based assay according to the manufacturer's instructions using the 10 µL viral aliquot.

2. Isolation and transduction of lineage-negative cells from mouse bone marrow (Figure 1A)

NOTE: Typically, to isolate enough cells, pairs of tibias, femurs, and humeri are harvested from each mouse. Pelvic and spinal bones may also be harvested as a source of lineage-negative cells.

1. Isolation of bone marrow cells
   1. Euthanize 8-10 week old male CRISPR/Cas9 knock-in mice by 5% isoflurane followed by cervical dislocation, then disinfect their skin with 70% ethanol.
   2. Using dissecting scissors, make a transverse incision in the skin just below the ribcage and peel the skin distally in both directions to expose the legs and arms.
   3. Carefully separate the lower limbs from the hip bone by dislocating the hip joint. Cut along the femur head to remove the femur completely from the hip. Dislocate the knee and cut at the joint to separate the femur and tibia, while keeping the bone epiphysis intact. Dislocate the ankle joint and peel away the foot and extra muscle.
   4. Using dissecting scissors, cut over the shoulder to detach the upper limbs. Dislocate the shoulder, then cut at the elbow joint to harvest the humerus bone.
   5. Use cellulose-fiber wipes to carefully remove muscles from the femurs, tibias, and humeri. Take extra precaution to ensure that the bones do not break during this process.
   6. Place the isolated bones into a 50 mL conical tube containing RPMI, and place on ice.
      NOTE: The following steps should be carried out in a biosafety class II cabinet.
   7. Transfer the bones into a sterile, 100 mm culture dish.
   8. Grasp the bone with blunt forceps, and using dissecting scissors, carefully cut both epiphyses.
      NOTE: An insufficient cutting will lead to an incomplete flush of bone marrow, while overly aggressive cutting will result in cell loss.
   9. Fill a 10 mL syringe with ice-cold RPMI, and using a 22 G needle, flush the bone marrow from the shaft into a new 100 mm culture dish.
      NOTE: Bones will become white and translucent if the bone shaft has been well-flushed. If not, re-cut the bone ends and flush again.
   10. After all the bone marrow has been collected, make a single-cell suspension by passing the bone marrow several times through a 10 mL syringe with an 18 G needle. Repeat 10x to ensure a single-cell suspension.
   11. Filter cell suspension through a 70 µm cell strainer into a 50 mL conical tube.
   12. Centrifuge at 310 x g for 10 min at 4 °C.
   13. Aspirate the supernatant and resuspend the cell pellets in an appropriate volume of optimized separation buffer for the following cell separation process.
2. Isolation and lentivirus transduction of lineage-negative cells
   ​NOTE: Mouse lineage-negative cells are isolated from the bone marrow of Cas9 transgenic mice³, or other strains of mice, using a lineage depletion kit according to the manufacturer's instructions. Typically, lineage-negative cells account for 2%-5% of whole bone marrow nucleated cells, and the purity is usually greater than 90% following isolation. The isolated lineage-negative cells are cultured in serum-free hematopoietic cell expansion medium supplemented with 20 ng/mL recombinant murine TPO and 50 ng/mL recombinant murine SCF, then transduced with the lentivirus vector for 16 h at a multiplicity of infection (MOI) = 100.
   1. To isolate lineage-negative cells, use the lineage cell depletion kit according to the manufacturer's instructions.
   2. After isolation resuspend the lineage-negative cells in 1 mL of serum-free hematopoietic cell expansion medium.
   3. Seed the cells into a 6 well plate at a density of 1.5 x 10⁶ cells/mL (5 x 10⁵ lineage-negative cells/mouse.)
   4. Add recombinant murine TPO and SCF into wells at final concentrations of 20 ng/mL and 50 ng/mL, respectively.
   5. Pre-incubate cells at 37 °C in 5% CO₂ for ~2 h.
   6. Add lentivirus at MOI = 100, 4 µg/mL polybrene, and penicillin/streptomycin to the wells and incubate at 37 °C, 5% CO₂ for 16-20 h (Figure 1B).
   7. On the following day, collect the lentivirus transduced cells into a 15 mL conical tube and centrifuge at 300 g for 10 min.
   8. Carefully aspirate the supernatant and resuspend the pellet in 200 µL of RPMI per mouse. Keep the cells at RT until transplantation into mice (section 3).

3. Transplantation of transduced cells into lethally irradiated mice

1. On the day of bone marrow transplantation, place recipient mice into an eight-slice pie cage and expose them to two doses of whole body irradiation (550 Rad/dose, total dose = 1100 Rad), with approximately 4 h between each irradiation session.
2. After the second irradiation session, inject transduced lineage-negative cells to each anesthetized recipient mouse via the retro-orbital vein plexus (200 µL in total) using an insulin syringe (Figure 1C).
3. After irradiation, mice should be housed in sterilized cages and provided with a soft diet and drinking water supplemented with antibiotics for 14 d.
4. At 3-4 weeks after bone marrow transplantation, analyze peripheral blood to check for the engraftment of transduced donor cells (section 4).

4. Evaluating the chimerism of peripheral blood

1. Anesthetize mice with 5% isoflurane and obtain a blood sample from a retro-orbital vein using capillary tubes, and collect it into K₂EDTA tubes (the volume in one capillary tube is sufficient for the following assay).
2. Transfer 20 µL of blood from the K2EDTA tubes into the 5 mL round bottom polystyrene test tubes, and put on ice.
3. Add 1.5 mL of RBC lysis buffer to lyse red blood cells. Incubate for 5 min on ice.
4. To neutralize the lysis buffer, wash samples with FACS buffer (1.5 mL/sample).
5. Centrifuge at 609 x g at r_max for 5 min at 4 °C. Discard the supernatant.
6. Incubate the cells with a cocktail of monoclonal antibodies (diluted in 100 µL FACS buffer/sample) at RT for 20 min in the dark. A complete list of antibodies is provided in the Materials section above.
7. Wash the cells once with FACS buffer (2 mL/sample). Centrifuge at 609 x g at r_max (1,800 rpm) for 5 min at 4 °C. Discard the supernatant completely.
8. Fix the cells with paraformaldehyde containing fixation buffer (100 µL/tube) for 10 min at 4 °C.
9. Wash cells once with FACS buffer (3 mL/sample). Centrifuge at 609 x g at r_max (1,800 rpm) for 5 min at 4 °C. Discard the supernatant completely.
10. Suspend the pellet in 400 µL of FACS buffer.
11. Keep the samples at 4 °C until analysis by flow cytometry.

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

...

Materials

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