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 preparation and storage are discussed elsewhere17,18. 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.
<ol>
	<li>Prepare a 1:200 solution of collagen (0.0005%) in 1x PBS.</li>
	<li>Coat a 6 well plate with collagen solution and incubate at 37 &#176;C, 5% CO2 for ~30 min.</li>
	<li>Seed 293T cells at a density of 1 x 106 cells per well and incubate at 37 &#176;C, 5% CO2 for ~2 h.</li>
	<li>To prepare the mixture of three transfection plasmids for one well, combine 0.9 &#181;g of lentivirus vector, 0.6 &#181;g of psPAX2, and 0.3&#160;&#181;g of pMD2.G, then achieve a total volume of 10 &#181;L 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.</li>
	<li>Carefully add 50 &#181;L of 1x PBS and 5 &#181;L of the diluted PEI MAX (1.0 mg/mL)&#160;to the plasmid mixture and incubate for 15 min at room temperature (RT) (Table 1).</li>
	<li>Add 1 mL of DMEM to the mixture.</li>
	<li>Aspirate media from the 6 well plate, add 1 mL of plasmid mixture, and incubate at 37 &#176;C, 5% CO2 for ~3 h.</li>
	<li>Replace the media with 2 mL of fresh DMEM and incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	<li>Add 1 mL of fresh DMEM and incubate at 37 &#176;C, 5% CO2 for an additional 24 h (total incubation time is 48 h).</li>
	<li>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.</li>
	<li>Filter the supernatant through a 0.45 &#181;m filter.</li>
	<li>Transfer the filtrate to polypropylene centrifuge tubes.</li>
	<li>Ultracentrifuge at 4 &#176;C and 72,100 x g at rmax for 3 h.</li>
	<li>Carefully aspirate the supernatant, leaving behind the white pellet.</li>
	<li>Resuspend the pellet with 100 &#181;L of serum-free hematopoietic cell expansion medium without aeration.</li>
	<li>Keep a 10 &#181;L aliquot to measure the viral titer and store all remaining aliquots at -80 &#176;C until required.</li>
	<li>Titrate the virus with a qPCR-based assay according to the manufacturer&#39;s instructions using the 10 &#181;L viral aliquot.</li>
</ol>
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.
<ol>
	<li>Isolation of bone marrow cells
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Transfer the bones into a sterile, 100 mm culture dish.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Filter cell suspension through a 70 &#181;m cell strainer into a 50 mL conical tube.</li>
		<li>Centrifuge at 310 x g for 10 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the cell pellets in an appropriate volume of optimized separation buffer for the following cell separation process.</li>
	</ol>
	</li>
	<li>Isolation and lentivirus transduction of lineage-negative cells 
	&#8203;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&#39;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.
	<ol>
		<li>To isolate lineage-negative cells, use the lineage cell depletion kit according to the manufacturer&#39;s instructions.</li>
		<li>After isolation resuspend the lineage-negative cells in 1 mL of serum-free hematopoietic cell expansion medium.</li>
		<li>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.)</li>
		<li>Add recombinant murine TPO and SCF into wells at final concentrations of 20 ng/mL and 50 ng/mL, respectively.</li>
		<li>Pre-incubate cells at 37 &#176;C in 5% CO2 for ~2 h.</li>
		<li>Add lentivirus at MOI = 100, 4 &#181;g/mL polybrene, and penicillin/streptomycin to the wells and incubate at 37 &#176;C, 5% CO2 for 16-20 h (Figure 1B).</li>
		<li>On the following day, collect the lentivirus transduced cells into a 15 mL conical tube and centrifuge at 300 g for 10 min.</li>
		<li>Carefully aspirate the supernatant and resuspend the pellet in 200 &#181;L of RPMI per mouse. Keep the cells at RT until transplantation into mice (section 3).</li>
	</ol>
	</li>
</ol>
3. Transplantation of transduced cells into lethally irradiated mice
<ol>
	<li>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.</li>
	<li>After the second irradiation session, inject transduced lineage-negative cells to each anesthetized recipient mouse via the retro-orbital vein plexus (200 &#181;L in total) using an insulin syringe (Figure 1C).</li>
	<li>After irradiation, mice should be housed in sterilized cages and provided with a soft diet and drinking water supplemented with antibiotics for 14 d.</li>
	<li>At 3-4 weeks after bone marrow transplantation, analyze peripheral blood to check for the engraftment of transduced donor cells (section 4).</li>
</ol>
4. Evaluating the chimerism of peripheral blood
<ol>
	<li>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).</li>
	<li>Transfer 20 &#181;L of blood from the K2EDTA tubes into the 5 mL round bottom polystyrene test tubes, and put on ice.</li>
	<li>Add 1.5 mL of RBC lysis buffer to lyse red blood cells. Incubate for 5 min on ice.</li>
	<li>To neutralize the lysis buffer, wash samples with FACS buffer (1.5 mL/sample).</li>
	<li>Centrifuge at 609 x g at rmax for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Incubate the cells with a cocktail of monoclonal antibodies (diluted in 100 &#181;L FACS buffer/sample) at RT for 20 min in the dark. A complete list of antibodies is provided in the Materials section above.</li>
	<li>Wash the cells once with FACS buffer (2 mL/sample). Centrifuge at 609 x g at rmax (1,800 rpm) for 5 min at 4 &#176;C. Discard the supernatant completely.</li>
	<li>Fix the cells with paraformaldehyde containing fixation buffer (100 &#181;L/tube) for 10 min at 4 &#176;C.</li>
	<li>Wash cells once with FACS buffer (3 mL/sample). Centrifuge at 609 x g at rmax (1,800 rpm) for 5 min at 4 &#176;C. Discard the supernatant completely.</li>
	<li>Suspend the pellet in 400 &#181;L of FACS buffer.</li>
	<li>Keep the samples at 4 &#176;C until analysis by flow cytometry.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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 
It is recommended to test gRNAs in vitro to assess editing efficiency prior to conduct in vivo experiments. The efficiency of gRNAs is tested using a cell-free in vitro transcription and screening system. The transcribed gRNA is validated by measuring its efficiency at cleaving the template DNA in the presence of recombinant Cas9 protein, using agarose gel electrophoresis. Commercially available kits are available for this purpose.
Here, indel mutations are characterized by the TA cloning of PCR products amplified from edited region, transforming bacterial cells with those plasmids, and picking up individual colonies for Sanger sequencing. However, this method is laborious and time-consuming. Alternatively, next-generation sequencing (NGS) or pooled DNA sequencing followed by TIDE analysis can be performed19. The TIDE algorithm was created to analyze Sanger sequence traces generated from complex samples. It has been shown that indel estimates with TIDE are typically consistent with those off-targeted NGS20. The analytical software is available online at &#60;http://tide.nki.nl&#62;.
Generation of high-titer lentivirus particles 
The viral vesicular stomatitis virus G-protein, which is essential for cell infection, is highly pH-sensitive. Thus, it is important to keep the culture medium within an acceptable pH range, and it should not develop a yellowish appearance. Collagen-coated dishes for virus generation are employed because it accelerates the attachment of HEK293T cells and allows the performance of transfection within a few hours, rather than waiting overnight. However, depending on the experimental schedule, overnight incubation can also be considered.
Purification of lentivirus particles 
To achieve efficient transduction of hematopoietic stem cells, it is necessary to generate high-titer lentivirus. Optimization of centrifugation speed is a key feature. While the concentration of lentivirus is usually performed at 90,000 x g, several reports have shown that virus recovery increases if the material is centrifuged at the lower speed of 20,000 x g18. The production of high-titer lentivirus preparation without ultracentrifugation has also been&#160;suggested17. It should be noted that it is important to suspend the virus centrifugation pellet while avoiding vigorous pipetting to minimize aeration and maintain virus integrity. High-titer lentivirus particles are required for efficient transduction of hematopoietic stem cells11. Pilot experiments revealed that an MOI of 100 is optimal with regards to transduction efficiency and cell viability. It is recommended to evaluate lentivirus stocks on the basis of cell viability and transduction efficiency.
Storage of lentivirus particles
The lentivirus titer is highly sensitive to temperature, and the titer can be drastically reduced by inappropriate storage conditions and repeated freeze-thaw cycles. It has been found that transduction efficiency of lentivirus decreases rapidly when stored at 4 &#176;C [t(1/2) = 1.3 days] or subjected to multiple freeze-thaw cycles [t(1/2) = 1.1 rounds]. It is recommended that the virus preparations be snap-frozen in liquid nitrogen or crushed dry ice soon after the virus pellet is suspended. The viral stocks should be maintained at -80 &#176;C and thawed on ice to RT just prior equilibration and use11.
Several potential limitations should be noted. First, the introduction of off-target indel mutations by CRISPR/Cas9 has long been appreciated. It has also been shown that CRISPR/Cas9 can induce off-target mutations in vivo21. In practice, off-target indel mutations can be avoided by using gRNA sequences that are closely matched to target genome sites and have more than four mismatches to predicted secondary sites. Such design can be done with existing in silico tools22. Other computational tools to predict gRNA with minimized off-target actions are available (&#60;http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design&#62; or &#60;http://www.Benchling.com&#62;). It may also be beneficial to analyze an animal model using two or more different gRNAs to confirm the phenotype and minimize the possibility that the observed phenotype is mediated by an off-target effect of a specific gRNA.
In addition to conventional indel mutations created by CRISPR/Cas9, larger deletions that extend beyond kilobases have been reported. This can confound studies; however, those larger deletions are reported to be much lower frequency compared to indels23. Another potential problem is genetic compensation. It has been reported that mutant RNA with a premature termination codon (PTC) can result in the upregulation of related genes with sequence similarity by COMPASS complex-mediated activation of transcription24,25. This event has been suggested to be a mechanism that can lead to phenotypic differences between knock-out and knockdown approaches of gene ablation. Because CRISPR/Cas9-mediated genome editing heavily relies on stochastic introduction of frame-shift mutations that lead to the generation of PTC, genetic compensation can modify the phenotype. To avoid genetic compensation, experiments can be considered in which a gene&#39;s regulatory sequences are targeted by CRISPR/Cas9 or by the introduction of epigenetic modifiers using Cas9 as an RNA-guided DNA recognition platform.
Lastly, it should be acknowledged that hematopoiesis from cells engrafted into lethally-irradiated mice may differ from the native conditions of hematopoiesis. Furthermore, irradiation can have systemic effects on the organism that may confound interpretation of experiments examining the consequences gene mutations in hematopoietic cells.
Researchers have taken advantage of catalytically inactive Cas9 (dCas9) proteins as a &#34;RNA-guided DNA recognition platform&#34; and used dCas9 fusion proteins to localize effector domains to specific DNA sequences to either repress (CRISPRi) or activate (CRISPRa) transcription off-target genes26,27. While this protocol uses catalytically active Cas9 transgenic mice to introduce dsDNA cleavage in genomic DNA sequence, epigenetic modification to repress or activate specific genes is applicable by fusing dCas9 with chromatin modifier domains such as dCas9-KRAB or dCas9-VP64, respectively. Alternatively, dCas9 can be used as a transcriptional repressor as its own, by blocking transcriptional machinery to access to the gene site27. More recently, Zhou et al. established dCas9-SunTag-p65-HSF1 (SPH) transgenic mice that express a modified version of an epigenetic activator fused with dCas9 and showed that this CRISPRa system is functional in vivo28.
Our lab predominantly uses this technology to study the role of clonal hematopoiesis in cardiovascular disease processes. In proliferating tissue, somatic mutations in cancer driver genes can confer a cellular growth advantage and lead to aberrant clonal expansions. In the hematopoietic system, this process is known as &#34;clonal hematopoiesis&#34;, and it results in situations in which a substantial fraction of an individual&#39;s leukocytes are replaced by mutant clones. There is a growing appreciation that aberrant clonal expansions accelerate cardiovascular disease, such as atherosclerosis and heart failure, and contribute to morbidity and all-cause mortality15,29.
Recently, a causal connection between several of these somatic mutations and cardiovascular disease has been documented, and aspects of the underlying mechanisms have been elucidated10,13,14. However, these somatic mutations probably represent the &#34;tip of the iceberg&#34;, as epidemiological studies have shown that many additional candidate genes are associated with clonal hematopoiesis and, potentially, increased cardiovascular disease mortality. Thus, a systematic, higher throughput evaluation of clonal hematopoiesis driver genes is required. Current studies of the causal connection of clonal hematopoiesis and cardiovascular disease are based on the analysis of mice with hematopoietic system-specific conditional transgenic (Mx1-Cre, Vav-Cre, etc.) or mice after bone marrow transplant. These strategies, however, need to establish new mouse colonies and may become a financial and physical burden for researchers. Thus, a cheaper and more rapid method than the conventional murine transgenic/knock-out approach employed in the past is warranted. Lentiviral vectors to transduce HSPC and CRISPR technologies to engineer mutations, as described in this manuscript, facilitate the study of clonal hematopoiesis and cardiovascular disease.
In addition to generating conventional knock-out locus, this method is applicable to the production of truncated mutated proteins. For example, researchers have successfully generated a hematopoietic-Ppm1d truncation, which is frequently seen in patients with clonal hematopoiesis, by introducing frameshift mutations with a gRNA targeting exon 6 of the Ppm1d gene30.

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.

<table><tbody><tr><td>RPMI Medium 1640 (1X)</td><td>Gibco</td><td>11875-093</td><td>Medium</td></tr><tr><td>Sulfamethoxazole and Trimethoprim injection</td><td>TEVA</td><td>0703-9526-01</td><td></td></tr><tr><td>1/2 cc LO-DOSE INSULIN SYRINGE</td><td>EXELINT</td><td>26028</td><td>general supply</td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216--</td><td>Cell line</td></tr><tr><td>APC-anti-mouse Ly6C (Clone AL-21)</td><td>BD Biosciences</td><td>560599</td><td>Antibodies</td></tr><tr><td>APC-Cy7-anti-mouse CD45R (RA3-6B2)</td><td>BD Biosciences</td><td>552094</td><td>Antibodies</td></tr><tr><td>BD Luer-Lok disposable syringes, 10 ml</td><td>BD</td><td>309604</td><td>general supply</td></tr><tr><td>BD Microtainer blood collection tubes, K2EDTA added</td><td>BD Bioscience</td><td>365974</td><td>general supply</td></tr><tr><td>BD Precisionglide needle, 18 G</td><td>BD</td><td>305195</td><td>general supply</td></tr><tr><td>BD Precisionglide needle, 22 G&amp;nbsp;</td><td>BD</td><td>305155</td><td>general supply</td></tr><tr><td>BV510-anti-mouse CD8a (Clone 53-6.7)</td><td>Biolegend</td><td>100752</td><td>Antibodies</td></tr><tr><td>BV711-anti-mouse CD3e (Clone 145-2C11)</td><td>Biolegend</td><td>100349</td><td>Antibodies</td></tr><tr><td>Collagen from calf skin</td><td>Sigma-Aldrich</td><td>9007-34-5</td><td>general supply</td></tr><tr><td>Corning Costar Ultra-Low Attachment Multiple Well Plate, 6 well&amp;nbsp;</td><td>Millipore Sigma</td><td>CLS3471</td><td>general supply</td></tr><tr><td>CRISPR/Cas9 knock-in mice</td><td>The Jackson Laboratory</td><td>028555</td><td>mouse</td></tr><tr><td>DietGel 76A</td><td>Clear H&lt;sub&gt;2&lt;/sub&gt;O</td><td>70-01-5022</td><td>general supply</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM) - high glucose</td><td>Sigma Aldrich</td><td>D6429</td><td>Medium</td></tr><tr><td>eBioscience 1X RBC Lysis Buffer</td><td>Thermo fisher Scientific</td><td>00-4333-57</td><td>Solution</td></tr><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Life Sciences</td><td>353003</td><td>general supply</td></tr><tr><td>Falcon 5 mL round bottom polystyrene test tube</td><td>Life Sciences</td><td>352054</td><td>general supply</td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>352098</td><td>general supply</td></tr><tr><td>Falcon 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate&amp;nbsp;</td><td>Life Science</td><td>353046</td><td>general supply</td></tr><tr><td>Fisherbrand microhematocrit capillary tubes</td><td>Thermo Fisher Scientific</td><td>22-362566</td><td>general supply</td></tr><tr><td>Fisherbrand sterile cell strainers, 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td>general supply</td></tr><tr><td>FITC-anti-mouse CD4 (Clone RM4-5)</td><td>Invitrogen</td><td>11-0042-85</td><td>Antibodies</td></tr><tr><td>Fixation Buffer</td><td>BD Bioscience</td><td>554655</td><td>Solution</td></tr><tr><td>Guide-it Compete sgRNA Screening Systems</td><td>Clontech</td><td>632636</td><td>Kit</td></tr><tr><td>Isothesia (Isoflurane) solution</td><td>Henry Schein</td><td>29404</td><td>Solution</td></tr><tr><td>Lenti-X qRT-PCR Titration Kit&amp;nbsp;</td><td>Takara</td><td>631235</td><td>Kit</td></tr><tr><td>Lineage Cell Depletion Kit, mouse</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>Kit</td></tr><tr><td>Millex-HV Syringe Filter Unit, 0.45 mm</td><td>Millipore Sigma</td><td>SLHV004SL</td><td>general supply</td></tr><tr><td>PBS pH7.4 (1X)</td><td>Gibco</td><td>10010023</td><td>Solution</td></tr><tr><td>PE-Cy7-anti-mouse CD115 (Clone AFS98)</td><td>eBioscience</td><td>25-1152-82</td><td>Antibodies</td></tr><tr><td>PEI MAX</td><td>Polysciences</td><td>24765-1</td><td>Solution</td></tr><tr><td>Penicillin-Streptomycin Mixture</td><td>Lonza</td><td>17-602F</td><td>Solution</td></tr><tr><td>PerCP-Cy5.5-anti-mouse Ly6G (Clone 1A8)</td><td>BD Biosciences</td><td>560602</td><td>Antibodies</td></tr><tr><td>pLKO5.sgRNA.EFS.tRFP&amp;nbsp;</td><td>Addgene</td><td>57823</td><td>Plasmid</td></tr><tr><td>pMG2D</td><td>Addgene</td><td>12259</td><td>Plasmid</td></tr><tr><td>Polybrene Infection/Transfection Reagent</td><td>Sigma Aldrich</td><td>TR-1003-G</td><td>Solution</td></tr><tr><td>Polypropylene Centrifuge Tubes</td><td>BECKMAN COULTER</td><td>326823</td><td>general supply</td></tr><tr><td>psPAX2&amp;nbsp;</td><td>Addgene</td><td>12260</td><td>Plasmid</td></tr><tr><td>RadDisk &amp;ndash; Rodent Irradiator Disk</td><td>Braintree Scientific</td><td>IRD-P M</td><td>general supply</td></tr><tr><td>Recombinant Murine SCF</td><td>Peprotech</td><td>250-03</td><td>Solution</td></tr><tr><td>Recombinant Murine TPO&amp;nbsp;</td><td>Peprotech&amp;nbsp;</td><td>315-14</td><td>Solution</td></tr><tr><td>StemSpan SFEM</td><td>STEMCELL Technologies</td><td>09600</td><td>Solution</td></tr><tr><td>TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coli</td><td>Thermo fisher Scientific</td><td>K457501</td><td>Kit</td></tr><tr><td>Zombie Aqua Fixable Viability Kit</td><td>BioLegend</td><td>423102</td><td>Solution&amp;nbsp;</td></tr></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 blood (Figure 2A,B). Blood is isolated from the retro-orbital vein and appropriate markers are used to determine the identity of each hematopoietic cell population (i.e., neutrophils, monocytes, T cells, etc.) (Figure 3A,B). Genomic DNA can be isolated from RFP-positive blood cells, and sections of the targeted site DNA can be amplified by PCR and subcloned into TA cloning vectors for sequence analysis. These plasmids are transduced into E. coli and the target site sequences are determined by Sanger sequencing (Figure 4). Alternatively, target site sequences can be determined by other methods, such as Sanger sequencing of the pooled genome followed by tracking of indels by decomposition (TIDE) analysis10. For the control condition, mice are typically transplanted with cells that are transduced with a lentivirus expressing non-targeting guide RNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59977/59977fig1.jpg" /> 
Figure 1: Schematic illustration of this protocol. (A) Isolation of lineage-negative bone marrow cells from Cas9-expressing mice (section 2.1). (B) Lentivirus transduction of lineage-negative cells (section 2.2). (C) Retro-orbital injection of transduced cells into lethally irradiated wild type mice (section 3). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59977/59977fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59977/59977fig2.jpg" /> 
Figure 2:&#160;Efficient lentiviral transduction of mouse bone marrow lineage-negative cells in vitro.&#160;(A) Flow cytometry analysis reveals successful transduction of lineage-negative cells. Analysis was performed after 7 days of in vitro culture. (B) On average, 75.7% of cells were transduced in this assay (n = 3). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59977/59977fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59977/59977fig3.jpg" /> 
Figure 3:&#160;Reconstitution of lethally-irradiated mouse bone marrow by transduced lineage-negative cells.(A) Flow cytometry analysis of mouse peripheral blood following reconstitution by hematopoietic stem cells that were (bottom) or were not (top) transduced with lentivirus expressing RFP. Neutrophils are defined as Ly6G+ and Ly6Chi&#160;monocytes as Ly6G- and Ly6C+, and B cells as CD45R+. (B) In these assays, an average of 94.8%, 93.5%, and 82.7% of cells are RFP+ in the neutrophil, Ly6Chi&#160;monocyte, and B cell populations, respectively (n = 8). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59977/59977fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59977/59977fig4.jpg" /> 
Figure 4:&#160;Evaluation of gene editing in transduced blood cells.(A) Example of gene editing showing sequencing results of mutated Dnmt3a locus in RPF-positive blood cells. Deletions are shown as red dashes and insertions are denoted with red letters. (B) Summary of mutations that were detected. (C) 69% (11/16 clones) showed out-of-frame/premature stop mutations. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59977/59977fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid</td>
			<td>Size (bp)</td>
			<td>Amount per well (&#181;g)</td>
			<td>Ratio</td>
		</tr>
		<tr>
			<td>pLKO5.0</td>
			<td>7700</td>
			<td>0.9</td>
			<td>2</td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>10668</td>
			<td>0.6</td>
			<td>1</td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>5822</td>
			<td>0.3</td>
			<td>1</td>
		</tr>
		<tr>
			<td colspan="2">PEI-max (stock: 100 mg/mL)</td>
			<td>5 &#181;L/well</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Amounts of plasmid and PEI-max used for transfection.

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 ...

lentiviral-crisprcas9-mediated-genome-editing-for-study-hematopoietic

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

12.1K Views.  University of Virginia School of Medicine. 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. 

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

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Cancer Research

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Genetics

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.