Name: lentiviral crispr/cas9-mediated genome editing for the study of hematopoietic cells in disease models
Uploaded: 2019-10-03T13:45:00
Description: Watch this Scientific Journal Video about Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models at JoVE.com

S. S. was supported by an American Heart Association postdoctoral fellowship 17POST33670076. K. W. was supported by NIH grants R01 HL138014, R01 HL141256, and R01 HL139819.

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: &#60;https://media.addgene.org/cms/files/Zhang_lab_LentiCRISPR_library_protocol.pdf)&#62;. Optimized methods for high-titer lentivirus prepara...

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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 
...

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 others1,2,3,4,5. 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 expertise6,7,8,9, 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 processes13,14,15, 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&#160;section 4.

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			<td height="21" style="height:21px;width:515px;">1/2 cc LO-DOSE INSULIN SYRINGE</td>
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			<td height="21" style="height:21px;">APC-anti-mouse Ly6C (Clone AL-21)</td>
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			<td height="21" style="height:21px;">BD Microtainer blood collection tubes, K2EDTA added</td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) - high glucose</td>
			<td>Sigma Aldrich</td>
			<td>D6429</td>
			<td>Medium</td>
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			<td height="21" style="height:21px;">eBioscience 1X RBC Lysis Buffer</td>
			<td>Thermo fisher Scientific</td>
			<td>00-4333-57</td>
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			<td height="21" style="height:21px;">Falcon 100 mm TC-Treated Cell Culture Dish</td>
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			<td height="21" style="height:21px;">Falcon 50 mL Conical Centrifuge Tubes</td>
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			<td height="21" style="height:21px;">Fisherbrand microhematocrit capillary tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-362566</td>
			<td>general supply</td>
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			<td height="21" style="height:21px;">Fisherbrand sterile cell strainers, 70 &#956;m</td>
			<td>Fisher Scientific</td>
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			<td height="21" style="height:21px;width:515px;">FITC-anti-mouse CD4 (Clone RM4-5)</td>
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			<td style="width:113px;">11-0042-85</td>
			<td>Antibodies</td>
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			<td height="21" style="height:21px;">Fixation Buffer</td>
			<td>BD Bioscience</td>
			<td>554655</td>
			<td>Solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Guide-it Compete sgRNA Screening Systems</td>
			<td style="width:159px;">Clontech</td>
			<td style="width:113px;">632636</td>
			<td>Kit</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Isothesia (Isoflurane) solution</td>
			<td>Henry Schein</td>
			<td>29404</td>
			<td>Solution</td>
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			<td height="21" style="height:21px;">Lenti-X qRT-PCR Titration Kit&#160;</td>
			<td>Takara</td>
			<td>631235</td>
			<td>Kit</td>
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			<td height="21" style="height:21px;">Lineage Cell Depletion Kit, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Kit</td>
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			<td>17-602F</td>
			<td>Solution</td>
		</tr>
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			<td>Plasmid</td>
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			<td>Peprotech</td>
			<td>250-03</td>
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			<td height="21" style="height:21px;">Recombinant Murine TPO&#160;</td>
			<td>Peprotech&#160;</td>
			<td>315-14</td>
			<td>Solution</td>
		</tr>
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			<td height="21" style="height:21px;">StemSpan SFEM</td>
			<td>STEMCELL Technologies</td>
			<td>09600</td>
			<td>Solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coli</td>
			<td style="width:159px;">Thermo fisher Scientific</td>
			<td>K457501</td>
			<td>Kit</td>
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			<td height="21" style="height:21px;">Zombie Aqua Fixable Viability Kit</td>
			<td>BioLegend</td>
			<td>423102</td>
			<td>Solution&#160;</td>
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	</tbody>
</table>

lentiviral crispr/cas9-mediated genome editing for the study of hematopoietic cells in disease models

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.

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...

Watch this Scientific Journal Video about Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models at JoVE.com

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

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.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

We've developed an efficient and economical method to establish mice with gene manipulation specifically in hematopoietic stem cells, Using genome editing technology and lentivirus-mediated transgene delivery systems. The advantage of this protocol is that we can create animal models harboring specific mutations in hematopoietic cells in a rapid and cost-effective manner compared to conventional mouse transgenic approaches. Demonstrating the procedure will be Ying Wang, a researcher from my laboratory.To generate lentivirus particles, start by coding a six well plate with collagen solution and incubating it at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. Then, seed 293T cells onto the plate and incubate it for another two hours. Meanwhile, prepare the mixture of three transfection plasmids by combining 0.9 micrograms of lentivirus vector, 0.6 micrograms of psPAX2 and 0.3 micrograms of pMD2.G and deionized to 10 microliters per well. Then, carefully add 50 microliters of 1X PBS and five microliters of diluted PEI MAX to the plasmid mixture, and incubate the mixture at room temperature for 15 minutes. After the incubation, add one milliliter of DMEM.Aspirate the media from the six well plate and add one milliliter of plasmid mixture to each well. Incubate the plate for another three hours. Replace the media with fresh DMEM and incubate for 24 hours.Add one milliliter of fresh DMEM and incubate an additional 24 hours. After total 48 hour incubation, transfer the supernatant to a 50 milliliter tube and centrifuge it at 3000 times G for 15 minutes to remove any free-floating cells. Filter the supernatant through a 0.45 micrometer filter and ultra-centrifuge it for three hours.Carefully aspirate the supernatant, leaving behind the white pellet. Re-suspend the pellet with 100 microliters of serum-free hematopoietic cell expansion medium without aeration. Keep a 10 microliter aliquot to measure the viral titer and store the rest of the suspension at minus 80 Celsius until ready to use.After isolating the bones, place them into a 50 milliliter conical tube with RPMI and place the tube on ice. In a bio-safety cabinet, transfer the bones into a sterile 100 millimeter culture dish. Then grasp a bone with blunt forceps, and use dissection scissors to carefully cut both epiphyses.Fill a 10 milliliter syringe with ice cold RPMI and use a 22 gauge needle to flush the bone marrow from the shaft into a new 100 millimeter culture dish. After all the bone marrow has been collected, make a single cell suspension by passing the bone marrow through a 10 milliliter syringe with an 18 gauge needle. Filter the cell suspension through a 70 micrometer cell strainer into a 50 milliliter conical tube.Then centrifuge the tube according to the manuscript directions and aspirate the supernatant. Re-suspend the cell pellets in an appropriate volume of optimized separation buffer. To isolate lineage negative cells, use a lineage cell depletion kit according to the manufacturer's instructions.Re-suspend the isolated lineage negative cells in serum-free hematopoietic cell expansion medium. Pre-incubate the cells at 37 degrees celsius in 5%carbon dioxide for two hours. Then add lentivirus according to the manuscript directions and leave the cells in the incubator for another 16 to 20 hours.The next day, collect the lentivirus-transduced cells in a 15 milliliter conical tube and centrifuge them at 300 times G for 10 minutes. According to NHA guidelines, it is important to handle lentivirus vectors at Reece Group level two. Precautions must be taken, based on facility regulations.Carefully aspirate the supernatant and re-suspend the pellet in 200 microliters of RPMI per mouse. Keep the cells at room temperature until transplantation into mice. After the second irradiation session, retro-orbitally inject transduced delineage negative cells into each anesthetized mouse using an insulin syringe.Three to four weeks after bone marrow transplantation, obtain blood samples from the retro-orbital vein using capillary tubes. Transfer 20 microliters of blood into round-bottom polystyrene test tubes and place on ice. After RBC lysis, incubate the cells with a cocktail of monoclonal antibodies in the dark for 20 minutes at room temperature.After incubation, wash, fix and centrifuge the cells and re-suspend the cell pellet in 400 microliters of FACS buffer. Keep the samples at four degrees Celsius until analysis by flow cytometry. Success of lineage negative cell transduction can be evaluated with flow cytometric detection of RFP.It has been demonstrated that after seven days of in vitro culture, on average, 75.7%of cells are transduced. Flow cytometry has also been used to analyze mouse peripheral blood after reconstitution with transduced and non-transduced hematopoietic stem cells. An average of 94.8%of neutrophils, 93.5%of monocytes, and 82.7%of B cells express RFP.Genomic DNA from the RFP-positive blood cells has been PCR amplified and sub-cloned for sequence analysis. Various mutations are detected and 69%of the clones show out of frame or premature stop mutations. To succeed with this procedure, preparation of high antivirus stock and optimized conditions for transduction and transplantation of hematopoietic cells are required.We have applied this technique to study the law of in cardio-vascular disease. But it is also useful to study hematological malignancy. This method enables us to study the functions of new types of genes in hematopoietic system in high efficient and high manner.We gather this over the availability of transgenic mice.

Research

JoVE Journal

Immunology and Infection

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