Name: genome engineering of primary human b cells using crispr/cas9
Uploaded: 2020-11-03T14:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

This work was funded by the Children&#8217;s Cancer Research Fund (CCRF) and NIH R01 AI146009 to B.S.M.

Leukapheresis samples from healthy donors were obtained from a local blood bank. All experiments described here were determined to be exempt for research by the Institutional Review Board (IRB) and were approved by the Institutional Biosafety Committee (IBC) at the University of Minnesota.
NOTE: All experiments were performed in compliance with the universal precaution for bloodborne pathogens, with sterile/aseptic techniques and proper biosafety level-2 equipment.
1. Prepare supplements for B-cell expansion medium
<ol>
	<li>Reconstitute CpG oligonucleotide to a concentration of 1 mg/mL.</li>
	<li>Reconstitute CD40 ligand (CD40L) to a concentration of 100 &#956;g/mL.</li>
	<li>Reconstitute recombinant human IL-10 (rhIL-10) to a concentration of 50 &#956;g/mL.</li>
	<li>Reconstitute recombinant human IL-15 (rhIL-15) to a concentration of 10 &#956;g/mL. 
	NOTE: Keep each supplement in small aliquots at -20 &#176;C to -80 &#176;C for up to 6 months.</li>
</ol>
2. Prepare basal medium
<ol>
	<li>Combine B-cell basal medium with 5% (v/v) media supplement for in vitro immune cell expansion (e.g., CTS Immune Cell SR) and 1% (v/v) penicillin and streptomycin.</li>
	<li>Filter-sterilize the basal medium using a 0.22 &#181;m filter adaptor into a sterilized bottle.</li>
	<li>Keep the basal medium at 4 &#176;C for up to 1 month.</li>
</ol>
3. Prepare B-cell expansion medium
<ol>
	<li>Transfer the required amount of the basal medium into a sterile container to culture the B cells at 5 &#215; 105 cells/mL.</li>
	<li>Supplement the basal medium with 1 &#956;g/mL CpG, 100 ng/mL CD40L, 50 ng/mL rhIL-10, and 10 ng/mL rhIL-15.</li>
	<li>Filter the B-cell expansion medium using a 0.22 &#181;m filter.</li>
	<li>Equilibrate the B-cell expansion medium in the tissue culture incubator at 37 &#176;C, 5% CO2 with humidity for at least 30 min before use. 
	NOTE: Prepare fresh B-cell expansion medium to use for one day. Do not prepare the B-cell expansion medium to use for multiple days. This media recipe encourages proliferation of memory B cells and activated primary human B cells.</li>
</ol>
4. Human B-cell purification and expansion
NOTE: Add 99&#8211;100% isopropyl alcohol to a temperature-controlled freezing container, following the manufacturer&#8217;s instruction, and chill the freezing container at 4 &#176;C before starting step 4.1.
<ol>
	<li>Isolate PBMCs from a leukaphoresis sample
	<ol>
		<li>Transfer a leukaphoresis sample (approximate 8&#8211;10 mL) to a sterile 50 mL conical tube.</li>
		<li>Bring up the volume to 35 mL with sterile 1x phosphate-buffered saline (PBS).</li>
		<li>Carefully layer a 35 mL leukaphoresis sample on 15 mL of density-gradient medium.</li>
		<li>Centrifuge at 500 &#215; g for 25 min without brake, remove the plasma layer without disturbing the buffy coat (PBMC layer), collect the PBMCs from the interface, and transfer to a new sterile 50 mL conical tube.</li>
		<li>Bring up the PBMCs to 50 mL with 1x PBS.</li>
		<li>Centrifuge at 500 &#215; g for 5 min without brake. Remove the supernatant without disturbing the PBMC pellet, which may appear red.</li>
		<li>Add 7 mL of ammonium-chloride-potassium lysis buffer, pipette 3 times to mix well, and incubate at room temperature (RT) for 3 min.</li>
		<li>Bring up the volume to 50 mL with 1x PBS.</li>
		<li>Centrifuge at 400 &#215; g for 5 min with low-resistance brake. Remove the supernatant without disturbing the pellet. The pellet should look pinkish or white. 
		NOTE: To continue to culture the freshly isolated B cells, prepare the B-cell expansion medium before starting B-cell isolation (step 4.2).</li>
	</ol>
	</li>
	<li>B-cell isolation from PBMCs using human primary B-cell negative isolation kit
	<ol>
		<li>Resuspend PBMCs in the isolation buffer to a concentration of 5 &#215; 107 cells/mL. 
		NOTE: If the total number of PBMCs is less than 5 &#215; 107 cells, scale down the volume of isolation buffer to maintain 5 &#215; 107 cells/mL. Minimum and maximum volumes of cell suspension are 0.25 mL and 8 mL, respectively.</li>
		<li>Transfer up to 8 mL (5 &#215; 107 cells/mL) to a sterile, polypropylene, round-bottom tube with cap.</li>
		<li>Add 50 &#956;L/mL of Cocktail Enhancer to the PBMCs.</li>
		<li>Add 50 &#956;L/mL of Isolation Cocktail to the PBMCs, cap the tube, and invert 2&#8211;3 times to mix.</li>
		<li>Incubate at RT for 5 min; at the 4th minute of incubation, vortex magnetic microbeads for at least 30 s.</li>
		<li>Transfer 50 &#956;L of magnetic microbeads per 1 mL of PBMCs, cap the tube, and invert 2&#8211;3 times to mix.</li>
		<li>Top up to 10 mL with isolation buffer and gently pipette up and down 2&#8211;3 times.</li>
		<li>Place the tube in a magnetic station and incubate at RT for 3 min.</li>
		<li>Hold the magnet and tube together, and in one motion, invert the magnet and tube together to pour the cell suspension into the new tube. Discard the old tube.</li>
		<li>Repeat step 4.2.8 (reduce the incubation time to 2 min) and pour the B-cell suspension into a clean conical tube.</li>
		<li>The enriched B cells are ready to use. If cells will be used immediately, continue to section 4.3 (Human B-cell expansion). Check the purity of the isolated B cells by flow cytometry (optional). If cells are to be frozen before use, continue to step 4.2.12.</li>
		<li>To freeze the B cells, centrifuge at 400 &#215; g for 5 min, and discard the supernatant without disturbing the pellet.</li>
		<li>Resuspend the cells in freezing medium at 107 cells/mL, and aliquot 1 mL/cryovial.</li>
		<li>Place the cryovial in the chilled freezing container, and store at -80 &#176;C overnight; then, transfer the frozen cryovial to a liquid nitrogen tank; keep frozen cells up to 1 year. 
		NOTE: Expected yield of isolated B cells is 2%&#8211;8% of the total PBMCs, with 95%&#8211;99% viability.</li>
	</ol>
	</li>
	<li>Human B-cell expansion 
	NOTE: If using freshly isolated B cells, skip steps 4.3.1&#8211;4.3.5. Count the cells and transfer the required number of cells into a sterile conical tube and continue with step 4.3.6.
	<ol>
		<li>Pre-warm fetal bovine serum (FBS) in a water bath prior to thawing the B cells. Prepare 20 mL of B-cell expansion medium, transfer to a T25 flask, and pre-equilibrate the medium in a tissue-culture incubator (at 37 &#176;C, 5% CO2, with humidity) at least 15 min before use.</li>
		<li>Thaw B cells in a 37 &#176;C water bath. While waiting, transfer 2 mL of pre-warmed FBS into a sterile 15 mL conical tube.</li>
		<li>After the B cells are completely thawed, immediately add 1 mL of pre-warmed FBS, drop-wise, into the sample. Incubate at RT for 1 min.</li>
		<li>Gently pipette to resuspend the sample and transfer the whole volume, dropwise, into a conical tube containing 2 mL of pre-warmed FBS.</li>
		<li>Bring up the volume to 15 mL with sterile 1x PBS, cap, and invert the tube gently 2&#8211;3 times.</li>
		<li>Centrifuge at 400 &#215; g for 5 min.</li>
		<li>Discard the supernatant without disturbing the cell pellet, resuspend the cell pellet with 1 mL of pre-equilibrated B-cell expansion medium, and count the cells. The total cell number should be approximately 107 cells.</li>
		<li>Transfer the cells into a flask containing 20 mL of the pre-equilibrated B-cell expansion medium. The final concentration of the cells should be approximately 5 x 105 cells/mL.</li>
		<li>Incubate the flask vertically in a tissue-culture incubator.</li>
		<li>Refresh the expansion medium completely every 2 days by transferring the whole volume of B cells in to a sterile conical tube and repeat steps 4.3.6&#8211;4.3.9. 
		NOTE: T25 flask can hold 10&#8211;20 mL of medium; T75 flask can hold up to 20&#8211;60 mL of medium.</li>
	</ol>
	</li>
</ol>
5. Primary human B-cell engineering
<ol>
	<li>Engineer B cells at 48 &#177; 2 h after expansion/activation for optimal results. Prepare the B-cell expansion medium, aliquot 1 mL of the medium into a 48-well tissue-culture plate, and pre-equilibrate in a tissue-culture incubator at least 15 min prior to use. 
	NOTE: When designing a CRISPR/Cas9 sgRNA for a gene of interest, follow the steps outlined below. 
	- Design sgRNAs using an online tool11. 
	- Design sgRNAs on an exon that is common to all isoforms of the protein. 
	- Order chemically modified sgRNA from reputable companies. 
	- sgRNA usually comes in lyophilized form; reconstitute sgRNA in sterile DNase/RNase-free Tris-EDTA (TE) buffer to a concentration of 1 &#956;g/&#956;L.</li>
	<li>Prepare CRISPR/Cas9 transfecting substrate by mixing 1 &#956;L (1 &#956;g/&#956;L) of chemically modified sgRNA with 1.5 &#956;L (1 &#956;g/&#956;L) of chemically modified Streptococcus pyogenes Cas 9 (S.p. Cas9) nuclease per transfection reaction. For control, use 1 &#956;L of TE buffer instead of sgRNA. 
	NOTE: When CD19 sgRNA is used for a gene KO experiment, see Figure 2 for results. When AAVS1 sgRNA is used for a gene KI experiment, see Figure 5 for results.
	<ul>
		<li>Keep all the components on ice.</li>
	</ul>
	</li>
	<li>Mix gently and transfer 2.5 &#956;L of CRISPR/Cas9 transfecting substrate per reaction into a tube of a 0.2 mL 8-tube strip; set aside at RT.</li>
	<li>Turn on a nucleofector (electroporator), and prepare transfection reagents as shown in Table 1. 
	NOTE: This is a good step for a pause, if necessary, by putting all reagents on ice. Remove all reagents from ice when ready to resume the experiment. When using S.p. Cas9 protein, the investigator MUST pre-complex CRISPR/Cas9-sgRNA ribonucleoprotein by mixing 1 &#956;g sgRNA with 5 &#956;g of S.p. Cas9 protein, avoiding any bubbles, and incubating the mixture at RT for at least 20 min before use for optimal results.</li>
	<li>Count and transfer 106 B cells per transfection reaction into a sterile conical tube.</li>
	<li>Bring up the volume to 15 mL with sterile 1x PBS, and centrifuge at 400 &#215; g for 5 min. While waiting, prepare the primary cell transfection reagent (Table 2) and set aside at RT.</li>
	<li>Discard the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend the cell pellet with 10 mL of sterile 1x PBS and centrifuge at 400 &#215; g for 5 min.</li>
	<li>Discard the supernatant completely without disturbing the cell pellet.</li>
	<li>Transfer 0.5 &#956;g of chemically modified GFP mRNA (as a transfection reporter) per 106 B cells to the cell pellet (optional).</li>
	<li>Resuspend the cell pellet with 20 &#956;L primary cell transfection reagent per 106 B cells; mix gently by pipetting 5&#8211;6 times. Transfer 20.5 &#956;L per transfection reaction into the 0.2 mL tube of the 8-tube strip containing 2.5 &#956;L of the CRISPR/Cas9 transfection substrate.</li>
	<li>Pipette up and down once to mix and transfer the entire volume (23 &#956;L) into a transfection cuvette. Cap and tap the cuvette on the bench gently to ensure that the liquid covers the bottom of the cuvette.</li>
	<li>Use human primary B-cell protocol on the nucleofector for transfection. 
	NOTE: The nucleofector (electroporator) can be placed and be used outside the tissue-culture hood. Cap the cuvette to ensure sterility.</li>
	<li>Rest the electroporated cells in the cuvette at RT for 15 min.</li>
	<li>Transfer 80 &#956;L of the pre-equilibrated B-cell expansion medium from the tissue culture plate into the transfection reaction in the cuvette. Place the cuvette in the tissue culture incubator for 30 min.</li>
	<li>Gently pipette a couple times to mix and transfer the whole volume of the sample from the cuvette to an appropriate well of a 48-well tissue-culture plate containing 1 mL of the B-cell expansion medium. The final concentration of the cells should be 106 cells/mL.</li>
	<li>If performing a gene KI experiment, transfer rAAV6 vector at 500,000 multiplicity of infection into the appropriate well containing electroporated cells (approximately 45 min post-electroporation). See example rAAV6 vector construct in Figure 4A. 
	NOTE: For example: A control sample will be electroporated without CRISPR/Cas9 or the rAAV6 vector. A vector-only sample will be electroporated without CRISPR/Cas9 and then be transduced with the rAAV6 vector. A KI sample will be electroporated with CRISPR/Cas9 and then be transduced with the rAAV6 vector. rAAV6 must contain homology arms up- and downstream of the targeted DSB site for HDR.</li>
	<li>Place the plate in a tissue culture incubator at 37 &#176;C and 5% CO2 with humidity.</li>
	<li>Count the cells and record viability at day 1 post-engineering.</li>
	<li>Refresh the B-cell expansion medium every 2 days by counting the cells and then transferring the whole volume of the cells into a clean 1.5 mL microcentrifuge tube. Centrifuge at 400 &#215; g for 5 min, and discard the supernatant without disturbing the pellet. Resuspend the cells with 100 &#181;L of fresh B-cell expansion medium, and transfer to a well of a 24-well tissue culture plate. Bring up the medium volume to achieve a final cell concentration at 5 &#215; 105 cells/mL. 
	NOTE: A 48-well plate can hold up to 1 mL medium/well; a 24-well plate can hold up to 2 mL medium/well; a 12-well plate can hold up to 4 mL medium/well, and a 6-well plate can hold up to 8 mL medium/well.</li>
	<li>Allow the engineered cells to expand for at least 5 days before performing downstream analyses such as for flow cytometry analysis, TIDE analysis, and junction PCR.</li>
</ol>

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A patent has been filed on the methods of making and using genome- edited B cells with M.J.J, K.L., and B.S.M. as inventors. B.S.M is a consultant for and owns stock in Immusoft Inc. Immusoft Inc has sponsored research in the lab of B.S.M.

Precise genome engineering in primary human B cells has been challenging until recently9,10. We had previously published protocols using CRISPR/Cas9 to engineer primary human B cells9. Here, we outline improved protocols for B-cell isolation, expansion, and engineering to allow for efficient KO of CD19 or for knocking-in EGFP.
Here we demonstrate that our expansion protocol allows the rapid expansion of...

B cells are a subgroup of the lymphocyte lineage derived from hematopoietic stem cells. They perform a critical role in the adaptive humoral immune system by producing large amounts of antibodies in response to immune challenges1. B cells are also precursors of memory B cells and the terminally differentiated, long-lived plasma cells, thereby providing lasting humoral immunity2. Plasma cells, in particular, are unique among immune cells in their ability to produce large amounts of a specific antibody while surviving for years or decades3. Additionally, the ease of isolation from peripheral blood makes the B-cell lineage an excellent candidate for novel cell-based therapies4.
Previously, random integration methods, such as those using lentiviral vectors or a Sleeping Beauty transposon, have been used to engineer B cells for transgene delivery and expression5,6,7,8. However, the non-specific nature of these approaches makes it difficult to study the biological functions of a specific gene in the B cells and carries an inherent risk of insertional mutagenesis and variable transgene expression and/or silencing in the therapeutic setting.
The CRISPR/Cas9 system is a powerful genome engineering tool that allows researchers to precisely edit the genome of various cells in numerous species. Recently, two groups, including our own, have successfully developed methods for ex vivo expansion and targeted genome engineering of primary human B cells9,10. We will describe the process of purifying primary human B cells from a leukaphoresis sample. After that, we will describe our updated protocol for B-cell expansion and activation of isolated B cells. We will then describe a process for knocking out cluster of differentiation 19 (CD19), a specific B-cell receptor and a hallmark of B cells, by electroporation to introduce CRISPR/Cas9 mRNA together with CD19 sgRNA into activated B cells.
Cas9 mRNA gets translated and binds to the CD19 sgRNA to form a CRISPR/Cas9-sgRNA ribonucleoprotein complex (RNP). Subsequently, sgRNA in the complex leads Cas9 to create double-strand break (DSB) at the target sequence on exon 2 of the gene. The cells will repair the DSB by &#8220;non-homologous end joining&#8221; by introducing or deleting nucleotides, leading to frameshift mutation and causing the gene to be knocked out. We will then measure the loss of CD19 by flow cytometry and analyze indel formation by tracking of indels by decomposition (TIDE) analysis.
We will then describe the process of using CRISPR/Cas9 together with a recombinant AAV6 vector (rAAV6, a donor template for homology-directed repair (HDR)) to mediate site-specific insertion of enhanced green fluorescent protein (EGFP) at the adeno-associated virus integration site 1 (AAVS1) gene. The AAVS1 gene is an active locus without known biological functions and an AAV viral integration site on the human genome; therefore, it is considered a &#8220;safe harbor&#8221; for genome engineering. Here, we report that expansion and activation of B cells allowed up to 44-fold expansion in 7 days of culture (Figure 1). Electroporation of B cells showed a slight reduction of overall cell health (Figure 2A) at 24 h post-transfection. Scatter plot analysis of the CD19 marker (Figure 2B) showed up to 83% reduction in the edited cells (Figure 2C).
TIDE analysis of the chromatographs (Figure 3A) revealed that the % indels was similar to the % protein loss by flow cytometry (Figure 3B). Flow cytometry analysis of the KI experiment showed that the cells that received AAV vector (Figure 4), together with RNP, expressed up to 64% EGFP-positive cells (Figure 5A) and later displayed successful integration by junction polymerase chain reaction (PCR) (Figure 5B). Cell counts showed that all samples quickly recovered within 3 days post-engineering (Figure 5C).

<table><tbody><tr><td>Alt-R S.p. Cas9 Nuclease V3 protein, 500 ug</td><td>IDT</td><td>1081059</td><td>smaller size is also available</td></tr><tr><td>Amaxa P3 primary cell 4D- Nucleofector X Kit S (32 RCT)</td><td>Lonza</td><td>V4XP-3032</td><td></td></tr><tr><td>Ammonium-chloride-potassium (ACK) lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>CleanCap chemically modified Cas9 mRNA</td><td>Trilink Biotechnology</td><td>L-7206-1000</td><td></td></tr><tr><td>CpG ODN 2006 (ODN 7909) 5 mg</td><td>Invivogen</td><td>TLRL-2006-5</td><td>different sizes available</td></tr><tr><td>Cryostor CS10, 100 mL</td><td>STEMCELL Technology</td><td>7930</td><td></td></tr><tr><td>CTS Immune Cell SR</td><td>Thermo Fisher Scientific</td><td>A2596101</td><td></td></tr><tr><td>EasySep human B cells isolation kit</td><td>STEMCELL Technology</td><td>17954</td><td></td></tr><tr><td>eBioscience fixable viability dye eFlour 780</td><td>eBiosciences</td><td>65-0865-14</td><td></td></tr><tr><td>Excellerate B cell media, Xeno-free</td><td>R&amp;amp;D Systems</td><td>CCM031</td><td>B-cell basal medium</td></tr><tr><td>Falcon 14 mL Polypropylene Round-bottom Tube</td><td>Corning</td><td>352059</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>R&amp;amp;D Systems</td><td>S11550</td><td>For thawing B cells only</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>GeneMate SnapStrip&amp;reg; 8-Strip 0.2 mL PCR Tubes with Individual Attached Dome Caps</td><td>BioExpress</td><td>T-3035-1 / 490003-692</td><td></td></tr><tr><td>Hyclone 0.0067M PBS (No Ca, No Mg) or 1x PBS</td><td>GE lifesciences</td><td>SH30256.01</td><td></td></tr><tr><td>Lonza 4D Nucleofector core unit</td><td>Lonza</td><td>AAF-1002B</td><td></td></tr><tr><td>Lonza 4D Nucleofector X unit</td><td>Lonza</td><td>AAF-1002X</td><td></td></tr><tr><td>Mega CD40 Ligand</td><td>Enzo Life Sciences</td><td>ALX-522-110-C010</td><td></td></tr><tr><td>Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562-1EA</td><td>For freezing cells</td></tr><tr><td>Pen/Strep 100X</td><td>Sigma-Aldrich</td><td>TMS-AB2-C</td><td></td></tr><tr><td>PerCP anti-human CD19 clone HIB19</td><td>biolegend</td><td>302228</td><td>smaller size is also available</td></tr><tr><td>rAAV6 SA-GFP pakaging ( with our SA-GFP cassette see Figure 4.)</td><td>Vigene Biosciences</td><td>N/A</td><td>large scale packaging, 1e13 GC/mL, 500 mL</td></tr><tr><td>Recombinant human IL-10 protein 250 ug</td><td>R&amp;amp;D Systems</td><td>217-IL-250</td><td>different sizes available</td></tr><tr><td>Recombinant human IL-15 protein 25 ug</td><td>R&amp;amp;D Systems</td><td>247-ILB-025</td><td>different sizes available</td></tr><tr><td>The Big Easy EasySep Magnet</td><td>STEMCELL Technology</td><td>18001</td><td>different sizes available</td></tr><tr><td>Tris-EDTA (TE) buffer</td><td>Fisher Scientific</td><td>BP2476100</td><td></td></tr></tbody></table>

genome engineering of primary human b cells using crispr/cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

The updated expansion and activation protocol enabled the rapid expansion of B cells up to 44-fold in 7 days (Figure 1; n =3 donors). In the KO experiment, the B-cell count using Trypan blue staining showed more than 80% viable cells with a slight reduction in cell recovery in both the control and the CD19 KO samples at 24 h post-electroporation (Figure 2A; p &#8805; 0.05, n = 3 donors). This result indicates that electroporation slightly affected overall B-cell...

Watch this Scientific Journal Video about Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 at JoVE.com

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

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Research

JoVE Journal

Biology

6.2K Views.  University of Minnesota. Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

Video: Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

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

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 ...

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 ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Immunology and Infection

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Gene Editing of Primary Rhesus Macaque B Cells

B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (&#60;4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.

We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies.

B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy ...

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 ...

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.

A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) ...