Name: genome engineering of primary human b cells using crispr/cas9
Uploaded: 2020-11-03T14:00:00
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....

<ol>
	<li>LeBien, T. W., Thomas, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+lymphocytes:+how+they+develop+and+function.">B lymphocytes: how they develop and function.</a> Blood. 112 (5), 1570-1580 (2008).</li><li>Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M., Corcoran, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+antibody-secreting+plasma+cells.">The generation of antibody-secreting plasma cells.</a> Nature Reviews Immunology. 15, 160-171 (2015).</li><li>Slifka, M. K., Antia, R., Whitmire, J. K., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immunity+due+to+long-lived+plasma+cells.">Humoral immunity due to long-lived plasma cells.</a> Immunity. 8 (3), 363-372 (1998).</li><li>Spriggs, M. 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=Recombinant+human+CD40+ligand+stimulates+B+cell+proliferation+and+immunoglobulin+E+secretion.">Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.</a> Journal of Experimental Medicines. 176 (6), 1543-1550 (1992).</li><li>Fusil, F., 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+lentiviral+vector+allowing+physiologically+regulated+membrane-anchored+and+secreted+antibody+expression+depending+on+B-cell+maturation+status.">A lentiviral vector allowing physiologically regulated membrane-anchored and secreted antibody expression depending on B-cell maturation status.</a> Molecular Therapy. 23 (11), 1734-1747 (2015).</li><li>Luo, X. 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=Engineering+human+hematopoietic+stem/progenitor+cells+to+produce+a+broadly+neutralizing+anti-HIV+antibody+after+in+vitro+maturation+to+human+B+lymphocytes.">Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes.</a> Blood. 113 (7), 1422-1431 (2008).</li><li>Mock, U., Thiele, R., Uhde, A., Fehse, B., Horn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+lentiviral+transduction+and+transgene+expression+in+primary+human+B+cells.">Efficient lentiviral transduction and transgene expression in primary human B cells.</a> Human Gene Therapy Methods. 23 (6), 408-415 (2012).</li><li>Heltemes-Harris, L. 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=Sleeping+Beauty+transposon+screen+identifies+signaling+modules+that+cooperate+with+STAT5+activation+to+induce+B-cell+acute+lymphoblastic+leukemia.">Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.</a> Oncogene. 35 (26), 3454-3464 (2016).</li><li>Johnson, M. J., Laoharawee, K., Lahr, W. S., Webber, B. R., Moriarity, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+primary+human+B+cells+with+CRISPR/Cas9+targeted+nuclease.">Engineering of primary human B cells with CRISPR/Cas9 targeted nuclease.</a> Scientific Reports. 8, 12144 (2018).</li><li>Hung, K. L., 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=Engineering+protein-secreting+plasma+cells+by+homology-directed+repair+in+primary+human+B+cells.">Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells.</a> Molecular Therapy. 26 (2), 456-467 (2018).</li><li>Cui, Y., Xu, J., Cheng, M., Liao, X., Peng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+CRISPR/Cas9+sgRNA+design+tools.">Review of CRISPR/Cas9 sgRNA design tools.</a> Interdisciplinary Sciences. 10 (2), 455-465 (2018).</li><li>Rees, H. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+editing:+precision+chemistry+on+the+genome+and+transcriptome+of+living+cells.">Base editing: precision chemistry on the genome and transcriptome of living cells.</a> Nature Reviews Genetics. 19 (12), 770-788 (2018).</li><li>Spiegel, A., Bachmann, M., Jurado Jim&#233;nez, G., Sarov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-based+knockout+pipeline+for+reverse+genetics+in+mammalian+cell+culture.">CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.</a> Methods. 164-165, 49-58 (2019).</li><li>Suzuki, T., Kazuki, Y., Oshimura, M., Hara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+for+simultaneous+or+sequential+integration+of+multiple+gene-loading+vectors+into+a+defined+site+of+a+human+artificial+chromosome.">A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.</a> PLoS One. 9, 110404 (2014).</li><li>Bak, R. O., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-mediated+integration+of+large+gene+cassettes+using+AAV+donor+vectors.">CRISPR-mediated integration of large gene cassettes using AAV donor vectors.</a> Cell Reports. 20 (3), 750-756 (2017).</li><li>Shirley, J. L., de Jong, Y. P., Terhorst, C., Herzog, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+viral+gene+therapy+vectors.">Immune responses to viral gene therapy vectors.</a> Molecular Therapy. 28 (3), 709-722 (2020).</li><li>Martino, A. T., Markusic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response+mechanisms+against+AAV+vectors+in+animal+models.">Immune response mechanisms against AAV vectors in animal models.</a> Molecular Therapy - Methods and Clinical Development. 17, 198-208 (2020).</li></ol>

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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