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&#38;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&#38;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&#174; 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&#38;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&#38;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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Biology

6.1K 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

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...