This work was supported by the Cancer Prevention and Research Institute of Texas (RP160283, RP140001, and R1201), the Gabrielle&#8217;s Angel Foundation for Cancer Research, the Edward P. Evans Foundation, and the NIH (DK092883, CA183252, CA125123, P50CA126752, CA193235, and DK107413). M.C.G. is supported by Baylor Research Advocates for Student Scientists. Flow cytometry was partially supported by the NIH (National Center for Research Resources grant S10RR024574, National Institute of Allergy and Infectious Diseases AI036211, and National Cancer Institute P30CA125123) for the Baylor College of Medicine Cytometry and Cell Sorting Core.

The protocol follows the guidelines of Baylor College of Medicine human ethics committee. All experimental procedures performed on mice are approved by Baylor College of Medicine Institutional Animal Care and Use Committee.
1. sgRNA Fwd Design
<ol>
	<li>Navigate to http://www.crisprscan.org/?page=track8 to begin designing sgRNAs of interest.</li>
	<li>Click on the &#34;Mouse&#34; or &#34;Human&#34; button depending on the cell type of interest.</li>
	<li>Enter the gene of interest into the UCSC search box and press go.</li>
	<li>Zoom in and move to the region of the gene (i.e. a specific exon) that is of interest to target. 
	Note: To achieve efficient gene disruption it is recommended targeting the earliest exon(s) present in all transcribed isoforms in your specific cell type.</li>
	<li>Ensure that the potential sgRNA target sequences are listed under a &#34;CRISPRscan predictions on genes (CDS)&#34; heading.</li>
	<li>Find and click on a target sequence with a relatively high score and few off-targets (highlighted in bright green). Copy the full sequence displayed under the &#34;Oligo sequence&#34; section and paste it into a text document. Add &#34;ATAGC&#34; to the 3&#39; end of the sequence. Once completed, place the order for the full-length sequence. 
	Note: For each target sequence, CRISPRscan8 provides an &#34;integrated&#34; forward sgRNA primer that includes the T7 promoter, the protospacer (target) sequence, and the universal scaffold overlap sequence (see Figure 1A). A full-length sequence is made of either 56 or 57 nucleotides depending on the length of protospacer.</li>
</ol>
2. sgRNA DNA Template Synthesis
<ol>
	<li>Dissolve and dilute the sgRNA Fwd and universal Rev primer (see Table 1 for the complete sequence). In order to obtain a 10 &#181;M final concentration, follow the manufacturer instruction on how to dilute primers to 100 &#181;M, then make a 1:10 dilution with nuclease-free water.</li>
	<li>To perform overlapping PCR (see Figure 1B), mix the following in PCR strip tubes: 2 &#181;L Forward sgRNA primer (10 &#181;M), 2 &#181;L Universal Rev scaffold primer (HPLC purified) (10 &#181;M), 10 &#181;L High fidelity DNA polymerase master mix (2x) and 6 &#181;L nuclease-free water.</li>
	<li>Run the PCR with the following program: 95 &#176;C for 3 min,98 &#176;C for 5 s, 60 &#176;C for 5 s, 72 &#176;C for 10 s, Go to 2 for 6 cycles, 72 &#176;C for 1 min, 4 &#176;C forever.</li>
	<li>Purify the PCR product using DNA purification columns (See Table of Materials). Follow manufacturer instructions and elute with 11.5 &#181;L of elution buffer.</li>
	<li>Measure the concentration of the PCR products on a spectrophotometer. Blank the instrument with elution buffer. 
	Note: The DNA concentration should be between ~50 and ~120 ng/&#181;L.</li>
</ol>
3. In Vitro Transcription of sgRNA
<ol>
	<li>Mix the following components in PCR strip tubes (reagents are provided in the RNA synthesis kit): 4 &#181;L of eluted DNA, 4 &#181;L of dNTPs, 1 &#181;L of 10x Reaction Buffer, and 1 &#181;L of T7 RNA polymerase enzyme mix.</li>
	<li>Incubate the samples at 37 &#176;C for at least 4 h.</li>
	<li>Apply the RNase cleaning agent to remove RNase from gloved hands.</li>
	<li>Bring each RNA sample up to a total volume of 50 &#181;L with nuclease-free water (first step of RNA purification following manufacturer instructions).</li>
	<li>Proceed with RNA purification following manufacturer instructions and elute in 50 &#181;L of kit-provided nuclease-free water.</li>
	<li>Measure the concentration of the eluted sgRNA on a spectrophotometer. Blank the instrument with nuclease-free water. 
	Note: The expected yield after purification is 50 - 80 &#181;g of RNA (i.e. concentration of 1.0 - 1.5 &#181;g/&#181;L).</li>
	<li>Use the purified sgRNA immediately or store in aliquots of 2 - 4 &#181;L at -80 &#176;C for the long-term.</li>
</ol>
4. HSPC Isolation and Culture
<ol>
	<li>Murine HSPCs isolation and culture 
	Note: Male and female Ubc-GFP mice (JAX004353) and Rosa26-LSL-tdTomato (JAX007914) crossed with Vav-iCre (JAX008610) at 2 - 6 months of age were used to obtain the results shown below.
	<ol>
		<li>Euthanize anesthetized mice through cervical dislocation. 
		Note: Two trained persons should independently verify successful euthanasia by noting a lack of respiration and heartbeat for at least 5 min. Remove the skin from the animals.</li>
		<li>Dissect tibias, femurs, and iliac crests of mice and remove all muscle and connective tissue around the dissected bones. Place intact bones into a tissue culture dish on ice with HBSS supplemented with 2% FBS (HBSS+). Move to a laminar flow hood as soon as al the bones have been cleaned and transferred to the tissue culture dish.</li>
		<li>Transfer cleaned bones to sterile mortar, containing 2 mL ice-cold HBSS+ per 3 bones. Using the pestle, crush bones into bone fragments, releasing marrow from within. Continue pestling until bones stop cracking under the pestle.</li>
		<li>Collect supernatant from the mortar and filter into a 50-mL conical tube using a 40 &#181;m cell strainer. Rinse the remaining bone fragments with 5 mL ice-cold HBSS+ and filter into the same 50 mL conical tube using the same 40 &#181;m strainer.</li>
		<li>Wash the cells twice by topping off the tube with ice-cold HBSS+ and centrifuging at 400 x g for 7 min. Discard the supernatant.</li>
		<li>Following the second wash resuspend the cell pellet in 20 mL of ice cold PBS. Count viable cells by trypan-blue exclusion9.</li>
		<li>Incubate 1 x 108 viable cells per 1 mL ice-cold HBSS+ with biotin-conjugated c-kit antibody at 1:100 dilution for 45 min on ice.</li>
		<li>Wash the cells with ice-cold HBSS+ by centrifuging at 400 x g for 7 min and discarding the supernatant.</li>
		<li>Incubate the cells with anti-biotin magnetic beads following manufacturer instructions.</li>
		<li>Isolate c-kit positive cells using magnetic columns or magnetic-activated cell sorters following manufacturer instructions. Collect c-kit positive cells in 15 mL conical tubes.</li>
		<li>Wash the cells twice with PBS by topping off the 15-mL conical tube and centrifuging at 400 x g for 7 min. Discard the supernatant. Between the two washes calculate the total number of viable cells by trypan blue exclusion9.</li>
		<li>Resuspend c-kit+ cells at a concentration of up to 106 viablecells per 100 &#181;L of culture medium for murine HSPCs supplemented with 2% FBS, 50 ng/mL mouse stem cell factor (SCF), 50 ng/mL mouse thrombopoietin (TPO), 10 ng/mL mouse IL-3, and 10 ng/mL mouse IL-6.</li>
		<li>Incubate the cells at 37 &#176;C, 5% CO2 for at least 1 h to a maximum of 24 h prior to the electroporation.</li>
	</ol>
	</li>
	<li>Human HSPCs isolation and culture 
	Note: These steps must be performed in a laminar flow hood.
	<ol>
		<li>Filter bone marrow/cord blood cells through a 40 &#181;m cell strainer.</li>
		<li>Dilute 1:2 the filtered bone marrow/cord blood cells with PBS supplemented with 1 mM EDTA (PBS/EDTA).</li>
		<li>Dispense 15 mL of density gradient medium into a 50-mL conical tube. Gently pour 30 mL of the diluted bone marrow/cord blood on the density gradient medium surface. If the volume of cell suspension is greater than 30 mL prepare multiple tubes. 
		Note: Make sure to keep the interface between the blood and the density gradient medium as sharp as possible.</li>
		<li>Spin the tubes at 750 x g for 30 min with no deceleration.</li>
		<li>Collect the mononuclear cell layer detectable at medium&#39;s interface and transfer to a clean 50 mL conical tube. Fill up the tube with PBS/EDTA and spin the cells at 280 g for 15 min. Discard the supernatant.</li>
		<li>Wash the cells twice in PBS/EDTA spinning at 400 x g for 7 min and discarding the supernatant.</li>
		<li>Incubate the mononuclear cells with anti-CD34 magnetic beads following manufacturer instructions.</li>
		<li>Isolate CD34+ cells using magnetic columns or magnetic-activated cell sorters following manufacturer instructions. Collect CD34+ cells in 15 mL conical tubes.</li>
		<li>Wash collected CD34+ cells twice by topping off the tube with PBS and spinning at 400 x g for 7 min and discarding the supernatant. Between the two washes calculate the total number of viable cells by trypan blue exclusion9.</li>
		<li>Resuspend the cells in culture medium supplemented with 100 ng/mL human SCF, 100 ng/mL human FLT3 ligand (FLT3L), 100 ng/mL human TPO at a concentration of ~2.5 x 105 viable cells/ml. Culture isolated CD34+ cells at 37 &#176;C, 5% CO2 for 24 h to a maximum 48 h prior to electroporation. 
		Note: Prior to electroporation check purity of enriched CD34+ population by flow cytometry.</li>
	</ol>
	</li>
</ol>
5. Electroporation
Note: The following protocol is optimized for 10 &#181;L electroporation tips. All steps should be performed in a laminar flow hood.
<ol>
	<li>Count cells by trypan blue exclusion9. Collect 2 x 105 viable cells per replicate (e.g. collect 6 x 105 cells for 3 electroporations).</li>
	<li>Wash cells twice with PBS, spinning at 300 x g for 7 min and carefully remove the supernatant.</li>
	<li>In parallel, prepare Cas9-sgRNA RNP complexes by incubating in PCR tubes for 20 - 30 min at RT 1.5 &#181;g Cas9 with 1 &#181;g of total sgRNA for each replicate, except the Cas9 only control. 
	Note: Cas9 protein is provided at 10 &#181;g/&#181;l concentration. To ensure accurate pipetting, dilute 1:10 the total amount of Cas9 protein needed with nuclease-free water (e.g for 10 electroporations take 1 &#181;l of Cas9 protein and dilute it with 9 &#181;l of nuclease-free water and incubate 1 &#181;l of diluted Cas9 per replicate. Depending on the concentration of the sgRNA, the total volume of Cas9 and sgRNA should be comprised between 1.7 and 2 &#181;l per replicate. If using two sgRNAs incubate 500 ng of each sgRNA per replicate; if using 3 sgRNAs incubate 330 ng of each sgRNA per replicate, and so on.</li>
	<li>Calculate the total volume of resuspension Buffer T needed by multiplying 10 &#181;L by the number of total replicates needed (e.g. 30 &#181;L for 3 replicates). Resuspend pelleted cells with the calculated volume of Buffer T. Ensure that the final concentration of the cell suspension is 2 x 107 cells/mL.</li>
	<li>Aliquot 10 &#181;L of resuspended cells/replicate into each PCR tube containing the pre-complexed sgRNA/Cas9 RNP (e.g. for a triplicate aliquot 30 &#956;L of cell suspension into a PCR tube containing pre-complexed Cas9-sgRNA).</li>
	<li>To set up the electroporation system turn on the instrument and add 3 mL of buffer E into an electroporation cuvette, and slide the cuvette into the cuvette holder.</li>
	<li>Set the desired electroporation conditions on device: human HSPCs (1600 V, 10 ms, 3 pulses) or mouse HSPCs (1700 V, 20 ms, 1 pulse). 
	Note: If desired, before performing experiments on CD34+ HSPCs, sgRNA efficiency can be tested on acute myeloid leukemia (AML) cell lines (e.g. HL60) using the same strategy (use antibiotic-free medium for AML cell lines) with the following electroporation conditions: Buffer R, 1350 V, 35 ms, 1 pulse.</li>
	<li>The electroporation is performed with a special electroporation pipette, which has a claw that latches onto the electroporation pipette tip. Hold the electroporation pipette, extend the claw, grab the disc inside the pipette tip, and then firmly press the pipette down to secure the tip.</li>
	<li>Pipette the sample up and down 10 times to mix. Take out 10 &#181;L, making sure there are no bubbles, and insert the pipette tip directly into the electrolytic buffer in the electroporation cuvette. Try not to touch the walls of the cuvette. Press &#34;start&#34; on the screen. After the &#34;complete&#34; message appears, remove the pipette, and dispense contents into well with new HSPC culture medium.</li>
	<li>Repeat for all samples. Change pipette tips between samples, only if necessary.</li>
</ol>

<ol>
	<li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337, 816-821 (2012).</li><li>Hsu, P. D., Lander, E. S., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157, 1262-1278 (2014).</li><li>Sternberg, S. H., Doudna, J. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+ablation+of+genes+in+human+hematopoietic+stem+and+effector+cells+using+CRISPR/Cas9.">Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.</a> Cell stem cell. 15, 643-652 (2014).</li><li>Gundry, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Genome+Editing+of+Murine+and+Human+Hematopoietic+Progenitor+Cells+by+CRISPR/Cas9.">Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.</a> Cell Rep. 17, 1453-1461 (2016).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. 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No conflict of interest to disclose

The RNP approach described in this detailed protocol enables efficient gene knockout in both mouse and human primary hematopoietic cells as well as in suspension cell lines. Although very high KO efficiencies are often obtained, several critical variables need to be considered. First, proper exon choice is key in guaranteeing that efficient indel incorporation corresponds to gene knockout. Two important factors related to exon choice are conservation across isoforms and exon position within transcripts. Isoform expression patterns are variable across cell types, so if gene KO across cell types is desired, exons that are invariant between cell types should be targeted. Isoform patterns can be verified using q-PCR or RNA-sequencing data. For exon position within transcripts, it is best to target early exons as frameshift mutations in the terminal or penultimate exon may escape nonsense-mediated decay10, producing proteins with novel or unknown functions rather than true nulls.
Determining the indel frequency is a critical step to estimate the KO efficiency and to correctly interpret the biological outcome of the experiment. Endonucleases assays (e.g. surveyor assay) have the advantage of being relatively fast and easy to perform. However, they tend to be inaccurate due to significant background signals and results are often difficult to reproduce. Moreover, they do not provide information on the quality of indels generated (e.g. length of insertions and deletions) in the experiment. Sanger sequencing provides a valuable alternative to endonuclease assays. Platforms such as TIDE11 (https://tide.nki.nl/) help to decompose sanger traces and produce accurate estimates of allelic disruption efficiency, while also providing the frequencies of individual indels. The major drawback is an absolute dependence on high-quality Sanger sequencing traces. High-throughput amplicon sequencing overcomes most of these issues. Amplicon libraries can be prepared starting with very low DNA inputs and the high coverage ensures reliable results. To reduce the cost of amplicon sequencing, we usually spike in amplicon libraries into larger sequencing runs (e.g. RNA-sequencing) using only 1 - 1.5% of the total reads. Finally, to confirm successful knockout, we strongly recommend assessing protein levels by western blots or flow cytometry, when possible.
As previously described, the method presented here is fast and straightforward and represents a new tool to study gene knock out in mouse and human HSPCs. While our protocol provides instructions for gene knockout with a tip-based electroporation system, other systems have been demonstrated to be highly effective12,13.
Two major limitations need to be acknowledged with respect to the RNP approach. First, as described by our group, the viability of HSPCs electroporated with in-vitro transcribed sgRNAs can be significantly compromised by electroporation, especially when conditions are not optimal6. If needed, commercially synthesized sgRNAs (i.e. eliminating in vitro transcription) may overcome this issue. These are becoming less expensive with time and can be purchased from several vendors. In this scenario, in vitro transcription could be used to generate a pool of sgRNA to screen for efficacy, and then the most efficient sgRNA(s) can be selected for synthesis. The second major limitation of the RNP approach is the inability to track successfully transfected cells, as in viral-based approaches. However, the high KO efficiency and rapid editing obtained with Cas9-sgRNA RNPs may outweigh these drawbacks in many experimental scenarios. We recommend using high-throughput amplicon sequencing to exactly determine the disruption efficiency in each experiment. If the knock-out of the gene of interest is expected to confer a proliferative phenotype (i.e. either reduced or increased proliferation compared to wild-type cells), determining the indel frequency at multiple time points allows one to assess whether KO cells undergo enrichment (i.e. indel frequency increases over time) or are outcompeted (i.e. indel frequency decreases over time).
Performing in vivo or in vitro experiments to understand the biological effects of loss of gene function requires the appropriate controls. As previously mentioned, in vitro transcribed sgRNAs may be toxic to cells, especially primary progenitor cells6. Additionally, DNA double-strand breaks caused by Cas9 protein can cause gene independent anti-proliferative response14. Therefore, we often use a number of control sgRNAs in CRISPR experiments and recommend a cell surface marker expressed on your cell type as well as a second target gene that is not expressed.
Short-term culture of human HSPCs in the presence of cytokines increases the gene disruption efficiency6 and is necessary to achieve high-efficiency KO. We have found 36 - 48 hours to be the ideal period of culture prior to electroporation6. Following electroporation, the cells can be further cultured keeping in mind that the longer the culture the higher the risk of losing multilineage engraftment capacity. Therefore, we usually perform transplants 6 hours after electroporation and never later than 24 hours after electroporation.
CRISPR/Cas9 has dramatically improved the ability of scientists to successfully edit the genome of mammalian cells. Making gene disruption more feasible in primary hematopoietic progenitor cells is predicted to rapidly enhance the scientific knowledge in the field of HSPCs and hematologic diseases. Importantly, the protocol described here makes it also possible to simultaneously generate multiple knockouts with high efficiency, allowing for the modeling of complex genetic landscapes, often seen in leukemic diseases.
Although the protocols described herein are focused on gene disruption, they may be easily adapted for gene knock-in experiments. This can be accomplished through the co-delivery of single-stranded oligonucleotide templates for homology-directed repair6,13 (HDR) or through the transduction of cells post-electroporation with AAV6 vectors containing the homology template12. The ability to repair or introduce specific mutations in HSPCs will facilitate new therapeutic strategies for gene therapy and the expanded study of cancer driver mutations in AML and other hematologic diseases. While HDR in human HSPCs has been successfull6,12,13, mouse HSPCs have been difficult to edit using this strategy6. Optimization of HDR protocols in human and particularly in mouse HSPCs will enable more specific editing and the development of tools to track edited cells using endogenous tagging.
Finally, it is also envisioned that disruption of mutant gain-of-function alleles could represent a potential therapeutic approach for congenital and acquired hematopoietic disorders. In this scenario, a DNA-free approach is advised in order to avoid possible integrations that could promote transformation. Moreover, the &#34;hit and run&#34; approach reduces the possibility of undesired off-target effects. More effort is needed in order to develop specific delivery systems that would open new avenues for the treatment of malignant and non-malignant hematologic diseases.

The advent of CRISPR/Cas9 has radically simplified gene editing in mammalian cells. Early CRISPR/Cas9 protocols utilized plasmid or virus-based delivery of Cas9 protein and sgRNA1,2,3. These approaches proved revolutionary for the development of cellular and animal models and have been also successfully applied to primary hematopoietic stem and progenitor cells (HSPCs)4,5. However, these methods have a number of drawbacks. First, gene disruption efficiency remains often below 50%4,5. While this is sufficient for generating knockout (KO) clones in cell lines, the necessity for short-term culture makes HSPCs not amenable to such a strategy. Second, the requirement for cloning limits the amount of sgRNAs that can be tested in single experiments and generates large, difficult to transfect constructs. Third, these approaches force scientists to slow turnaround times.
In order to overcome these limitations, our group developed and recently published an alternative CRISPR/Cas9 approach in HSPCs using Cas9-sgRNA ribonucleoproteins (RNPs)-based delivery6. In this strategy, the raw components of CRISPR (Cas9 protein and in vitro transcribed sgRNA) are pre-complexed and directly delivered into target cells via electroporation (Figure 1). As the half-life of the Cas9-sgRNA RNP complex is shorter than the time that plasmid or viral nucleic acid is transcribed, the off-target rate is lower compared to early approaches7. Moreover, the RNP approach adds the benefit of eliminating any source of exogenous DNA, which can randomly integrate into the target cell genome leading to cellular transformation.
This protocol is based on a streamlined workflow for RNP-based gene disruption experiments, as represented in Figure 1. The first step is designing and ordering primers for each sgRNA. These primers are utilized to make sgRNA DNA templates that are used for in vitro transcription (IVT) to obtain the sgRNAs. Purified sgRNAs are then incubated with previously purchased Cas9 protein, to form Cas9-sgRNA RNP complexes. Finally, pre-complexed Cas9-sgRNA RNPs are electroporated into cells. Following electroporation, editing efficiency can be tested and in vitro/in vivo experiments can be started, depending on needs. Below a detailed description of this innovative experimental approach can be found.

<table><tbody><tr><td>Tissue culture plates according to cell numbers</td><td></td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf microtubes</td><td>Sigma</td><td>Z606340-1000EA</td><td></td></tr><tr><td>50ml Falcon tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Nuclease Free Water - DEPC treated</td><td>ThermoFisher Scientific</td><td>AM9915G</td><td></td></tr><tr><td>KAPA HiFi HotStart ReadyMix (2X)</td><td>KAPA Biosystems</td><td>KK2600</td><td></td></tr><tr><td>MinElute PCR purification Kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>RNAse Zap RNAse Decontamination Solution</td><td>ThermoFisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>RNA Clean and Concentrator-25</td><td>Zymo</td><td>R1017</td><td></td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit</td><td>New England Biolabs</td><td>E2040S</td><td></td></tr><tr><td>Alt-R S.p. Cas9 Nuclease 3NLS, 100 &amp;micro;g</td><td>IDT</td><td>1074181</td><td></td></tr><tr><td>40 mm Cell Strainers</td><td>VWR</td><td>352340</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>GenDEPOT</td><td>CA008-050</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Regular</td><td>Corning</td><td>35010CV</td><td></td></tr><tr><td>AutoMACS Pro Separator or MACS manual Cell Separation columns</td><td>Miltenyi</td><td></td><td></td></tr><tr><td>Neon Transfection System Device</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Neon Transfection System 10mL kit</td><td>ThermoFisher Scientific</td><td>MPK1025</td><td>For larger numbers of cells (see protocol) Neon Transfection System 100 mL kit (# MPK10025) can be used</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>UltraPure 0.5M EDTA, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575020</td><td>For human experiments only</td></tr><tr><td>Lymphoprep</td><td>Stem Cell Technologies</td><td>7851</td><td>For human experiments only</td></tr><tr><td>CD34 MicroBead Kit UltraPure human</td><td>Miltenyi Biotec</td><td>130-100-453</td><td>For human experiments only</td></tr><tr><td>Stem Span SFEM II</td><td>Stem Cell Technologies</td><td>9605</td><td>For human experiments only</td></tr><tr><td>Recombinant Human SCF</td><td>Miltenyi Biotec</td><td>130-096-695</td><td>For human experiments only</td></tr><tr><td>Recombinant Human TPO</td><td>Miltenyi Biotec</td><td>130-094-013</td><td>For human experiments only</td></tr><tr><td>Recombinant Human FLT3L</td><td>Miltenyi Biotec</td><td>130-096-479</td><td>For human experiments only</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Mouse dissection tools</td><td></td><td></td><td>For mouse experiments only</td></tr><tr><td>Mortar and Pestle</td><td></td><td></td><td>For mouse experiments only</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14170112</td><td>For mouse experiments only</td></tr><tr><td>Biotin-conjugated anti c-kit antibody</td><td>eBioscience</td><td>13-1171-82</td><td>For mouse experiments only</td></tr><tr><td>Anti-Biotin MicroBeads</td><td>Miltenyi Biotec</td><td>130-090-485</td><td>For mouse experiments only</td></tr><tr><td>X VIVO-15, with L-Glutamine, Genatmycin and Phenol Red</td><td>Lonza</td><td>04-418Q</td><td>For mouse experiments only</td></tr><tr><td>Mouse IL-6</td><td>Peprotech</td><td>216-16</td><td>For mouse experiments only</td></tr><tr><td>Mouse TPO</td><td>Peprotech</td><td>315-14</td><td>For mouse experiments only</td></tr><tr><td>Mouse IL-3</td><td>Peprotech</td><td>213-13</td><td>For mouse experiments only</td></tr><tr><td>Mouse SCF</td><td>Peprotech</td><td>250-03</td><td>For mouse experiments only</td></tr></tbody></table>

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.

The goal of this protocol is to perform gene knockout (KO) in mouse and human HSPCs using Cas9-sgRNA RNPs. The workflow is easy and fast and allows for completion of experiments in less than a week from experimental design to assessment of KO efficiency.
Compared to plasmid- or virus-based approaches, the success of our protocol builds upon the combination of sgRNA RNPs and electroporation. This cloning-free strategy significantly shortens the time needed to obtain the components to transfect. Moreover, electroporation allows for transfection of up to 95% of cells, maximizing the delivery of the RNPs. sgRNAs can be synthetized in the lab through in vitro transcription (IVT), while purified Cas9 protein can be purchased from several companies (see protocol). sgRNA DNA templates for IVT are obtained performing a short PCR using the sgRNA Fwd primer (Figure 1A, B) and the universal scaffold Rev primer (Figure 1B - see Protocol). The PCR product is then used as a template for the IVT (Figure 1C). Cas9-sgRNA RNP complexes are generated incubating for 15 - 30 minutes the sgRNAs and the Cas9 (Figure 1C). Finally, sgRNA RNPs are electroporated into cells. Edited HSPCs can then be used to perform both in vitro and in vivo experiments.
Gene disruption in mouse cells 
To demonstrate the efficacy of the Cas9-sgRNA RNP electroporation strategy, c-kit+ HSPCs isolated from mice ubiquitously expressing GFP or tdTomato have been electroporated with sgRNA against GFP or tdTomato. Among the three sgRNA designed against GFP, GFP sg1 performed most efficiently, exhibiting approximately 70 - 75% disruption of GFP expression as determined by flow cytometry (Figure 2A, B). To quantify the gene disruption frequency at the genomic level, genomic DNA from the electroporated cells was isolated and T7 endonuclease I-based (T7EI) assay was performed. As shown in Figure 2C, T7EI assay detected up to 60% indel formation. Note that T7EI assays tend to underestimate the frequencies of indels. We also generated sgRNA against tdTomato and electroporated HSPCs expressing tdTomato from the Rosa26 locus. As shown in Figure 2D, tdTomato sg1 efficiently ablated the expression of tdTomato.
Gene disruption in human cells
Here, efficient disruption obtained with a single sgRNA approach targeting CD45, a cell surface marker expressed on hematopoietic cells, is shown. Three sgRNAs targeting different exons of CD45 were designed (CD45 sg1, sg2, and sg3). The efficiency of the sgRNAs was tested in the HL60 AML cell line (Figure 3A). Protein KO was assessed by flow cytometry 5 days after electroporation. CD45 sg1 was the most efficient (98% KO efficiency - Figure 3A) and was used for further experiments. Figure 3B shows CD45 KO in primary human CD34+ progenitor cells using CD45 sg1, assessed by flow cytometry 5 days after electroporation. While CD34 expression was unaffected, CD45 expression was lost in about 80% of the cells. 200.000 edited primary CD34+ cells were retro-orbitally transplanted into NSG mice 6 hours after electroporation. Bone marrow and spleen cells were harvested 3 months after transplant. Edited cells (HLA-ABC positive, CD45 negative, highlighted in red) are detectable only in sgRNA treated samples and not in Cas9 only controls (Figure 3C).
If desired, two sgRNAs targeting nearby sequences can be used to generate deletions. This approach allows for a fast estimate of the editing efficiency with a PCR and often produces higher disruption efficiency. Figure 3D shows the efficient generation of a 58 bp deletion in exon 10 of DNMT3A. 200.000 edited CD34+ cells were transplanted into NSG mice 6 hours after electroporation. The 58 bp deletion was clearly detected in the peripheral blood of engrafted NSG mice 5 months after injections (Figure 3E) confirming the editing of CD34+ cells with long-term engraftment capabilities.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57278/57278fig1.jpg" /> 
Figure 1: The fast and easy approach to generate sgRNA RNPs. A) Representation of the sgRNA Fwd primer. The sgRNA Fwd primer is a DNA oligonucleotide containing the T7 promoter, the protospacer sequence and an overlap sequence with the sgRNA scaffold. At the 3&#39; end, highlighted in red, is the &#34;ATAGC&#34; sequence that needs to be added to the sgRNA Fwd primer sequence obtained on CRISPRscan. B) Schematic representation of the overlap PCR performed to obtain the IVT template. The overlap between the sgRNA Fwd primer and the Universal Scaffold Rev primer allows for the extension and amplification by PCR. C) Cartoon describing the IVT reaction and the sgRNA RNP pre-complexing. The PCR product is purified and used as a template for the IVT. IVT generates a high amount of sgRNAs that can be used in multiple replicates (see protocol). sgRNA RNPs are generated incubating the sgRNA purified from IVT and the previously purchased Cas9 protein. <a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig1large.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/57278/57278fig2.jpg" /> 
Figure 2: Efficient gene disruption in mouse hematopoietic progenitor cells. A) An sgRNA targeting GFP (GFP sg1) together with Cas9 protein was electroporated into murine c-kit+ HSPCs expressing GFP, and analyzed by flow cytometry 24 hours after electroporation. B) Different amount of GFP sg1 was complexed with 1 &#181;g of Cas9 protein and electroporated. As little as 200 ng of GFP sg1 efficiently ablated GFP expression. C) T7EI assay performed on genomic DNA isolated from cells used in A-B. PCR amplicon spanning the Cas9-sgRNA cleavage site was diluted 1:4 in 1x Buffer 2 and hybridized slowly in a thermal cycler. Hybridized fragments were then digested with 1.25 U of T7 endonuclease I for 10 minutes at 37 &#176;C. Digested fragments were separated by polyacrylamide gel electrophoresis. Band intensities were analyzed using ImageJ software by plotting band intensities of each lane. % cleavage was calculated by the ratio of the intensities of the cleaved bands to uncleaved bands. D) c-kit+ HSPCs isolated from mice expressing tdTomato was electroporated with Cas9 protein complexed with sgRNA against tdTomato. Flow cytometry performed 24 hours after electroporation revealed that approximately 74% of cells deleted tdTomato. R26 = Rosa26. * p &#60;0.05, ** p &#60;0.01, *** p &#60; 0.001. This figure has been adapted from Gundry et al.6 <a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig2large.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/57278/57278fig3.jpg" /> 
Figure 3: Efficient gene disruption in human hematopoietic cells. A) Three sgRNAs targeting CD45 (CD45 sg1, sg2, and sg3) were designed and tested in the HL60 AML cell line (see protocol for electroporation conditions). Flow cytometry was performed 5 days after electroporation. CD45 sg1 was selected as the most efficient among the three sgRNAs (KO efficiency 98%). B) CD45 KO in primary human cord blood CD34+ HSPCs using CD45 sg1. CD45 loss can be detected in roughly 80% of the cells. Note that CD34 expression is unchanged after editing. C) Edited human HSPCs can be efficiently engrafted into NSG mice. Human nucleated cells display the normal HLA+/CD45+ phenotype in Cas9-only engrafted mice, whereas in mice engrafted with CD45-sg1 treated CD34+ cells, both HLA+/CD45+ and HLA+/CD45- populations are observed indicating engraftment of human CD45 KO cells. D) 2% agarose gel showing a 58 bp deletion generated by 2 sgRNAs (dual guide approach) in exon 10 of DNMT3A in human HSPCs from 3 different cord blood (CB1, CB2, and CB3). PCR was performed 3 days after the electroporation. E) 2% agarose gel demonstrating the persistence of the 58 bp deletion 5 months after transplantation into NSG mice. This figure has been adapted from Gundry et al.6 <a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Oligonucleotide</td>
			<td colspan="3">Sequence</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>Universal Rev scaffold primer&#160;</td>
			<td colspan="3">agcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac</td>
			<td>Universal reverse primer for overlap PCR</td>
		</tr>
		<tr>
			<td>hCD45 sgRNA 1</td>
			<td colspan="3">taatacgactcactataGGTGCTGGTGTTGGGCGCACgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>hCD45 sgRNA 2</td>
			<td colspan="3">taatacgactcactataGGGAGCAAGTGAGGATCCTCgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>hCD45 sgRNA 3</td>
			<td colspan="3">taatacgactcactataGGGATGCTTGTTCCCTTCAGgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>hDNMT3A Ex10 sg1</td>
			<td colspan="3">taatacgactcactataGGACACTGCCAAGGCCGTGGgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>hDNMT3A Ex10 sg2</td>
			<td colspan="3">taatacgactcactataGGGTGGCCAGCAGCCGCGCGgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>Rosa26 sgRNA</td>
			<td colspan="3">taatacgactcactataGGACACTGCCAAGGCCGTGGgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>GFP sg1</td>
			<td colspan="3">taatacgactcactataGGGCGAGGAGCTGTTCACCGgttttagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
		<tr>
			<td>tdTomato sg1</td>
			<td colspan="3">taatacgactcactataGGGGTACAGGCGCTCGGTGGgtttaagagctagaaatagc</td>
			<td>sgRNA Fwd primer sequence for overlap PCR</td>
		</tr>
	</tbody>
</table>
Table 1: Complete sequences of sgRNA Fwd primers used in the experiments and Universal Rev primer.

Watch this Scientific Journal Video about Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9 at JoVE.com

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

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. ...

highly-efficient-gene-disruption-murine-human-hematopoietic

Research

JoVE Journal

Genetics

13.4K Views.  Baylor College of Medicine. Een protocol voor snelle CRISPR/Cas9-gemedieerde gene verstoring van muis en mens primaire hematopoietische cellen wordt beschreven in dit artikel. Cas9-sgRNA ribonucleoproteins worden ingevoerd via electroporation met sgRNAs gegenereerd via in vitro transcriptie en commerciële Cas9. Hoge bewerken rendementen worden bereikt met beperkte tijd en de financiële kosten.

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

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

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

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

A Guide to Precise Gene Disruption in Primary Bone Marrow-Derived Macrophages

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the ...

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. ...

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.

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 ...

Immunology and Infection

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.

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

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