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

这项工作由儿童癌症研究基金（CCRF）和NIH R01 AI146009到B.S..M资助。

从健康献血者那里获得的白血病样本是从当地血库获得的。此处描述的所有实验均被确定为豁免供机构审查委员会 （IRB） 研究，并经明尼苏达大学机构生物安全委员会 （IBC） 批准。
注：所有实验都是按照血液传播病原体的普遍预防措施进行的，采用无菌/无菌技术和适当的生物安全2级设备。
1. 为B细胞扩张介质准备补充剂
<ol><li>将 CpG 寡核苷酸重组为?...

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直到最近9月10日，人类B细胞的精确基因组工程一直具有挑战性。我们以前曾发布过使用CRISPR/Cas9来设计原始人类B细胞9的协议。在这里，我们概述了改进的B细胞隔离，扩展和工程协议，以允许高效的KO CD19或敲击EGFP。
在这里，我们证明，我们的扩展协议允许在培养中快速扩展B细胞，在7天内扩展高达44倍（<st...

B细胞是来自造血干细胞的淋巴细胞系的子群。他们在适应性幽默免疫系统中发挥关键作用，产生大量的抗体来应对免疫挑战1。B细胞也是记忆B细胞和最终分化的长寿血浆细胞的前体，从而提供持久的幽默免疫力2。特别是等离子体细胞，在免疫细胞中独一无二，它们能够产生大量的特定抗体，同时存活多年或几十年。此外，从外周血液隔离的便利性使B细胞血统成为新细胞疗法4的极好候选者。
以前，随机整合方法，如那些使用扁桃体载体或睡美人转子，已经被用来工程B细胞的转基因传递和表达5，6，7，8。然而，这些方法的非特异性使得研究B细胞中特定基因的生物功能变得困难，并具有在治疗环境中插入突变和可变转基因表达和/或沉默的固有风险。
CRISPR/Cas9系统是一个强大的基因组工程工具，使研究人员能够精确编辑许多物种中各种细胞的基因组。最近，包括我们自己的两组已经成功地开发出了原始人类B细胞9、10的外活扩张和靶向基因组工程方法。我们将描述从白血病样本中净化原人类B细胞的过程。之后，我们将描述我们更新的 B 细胞扩展和激活孤立 B 细胞的协议。然后，我们将描述一个通过电波将CRISPR/Cas9 mRNA与CD19 sgRNA一起引入激活的B细胞，从而敲除分化19（CD19）的簇、特定的B细胞受体和B细胞的标志的过程。
Cas9 mRNA 被翻译并绑定到 CD19 sgRNA，以形成 CRISPR/Cas9-sgRNA 核糖核蛋白复合物 （RNP）。随后，复合物中的sgRNA导致Cas9在基因exon2上的目标序列中产生双链断裂（DSB）。细胞将通过引入或删除核苷酸来修复DSB，导致帧移位突变并导致基因被消灭。然后，我们将用流细胞测量测量CD19的损失，并通过分解（TIDE）分析对英德尔的形成进行跟踪分析。
然后，我们将描述使用CRISPR/Cas9与重组AAV6载体（rAAV6，同源定向修复（HDR）的捐赠模板）一起，以调解在腺相关病毒集成站点1（AAVS1）基因中插入增强型绿色荧光蛋白（EGFP） 的现场特定插入过程。AAVS1基因是一种没有已知生物功能的活性球体，也是人类基因组上的AAV病毒整合站点：因此，它被认为是基因组工程的"安全港"。在这里，我们报告说，B细胞的膨胀和激活允许在7天的培养中扩大44倍（图1）。B细胞的电扩散显示，在24小时后，整体细胞健康（图2A）略有下降。对 CD19 标记（图 2B）的分散图分析显示编辑的单元块（图 2C）减少高达83%。
TIDE对色谱仪（图3A）的分析显示，英德尔的百分比与流动细胞仪（图3B）所导致的蛋白质损失相似。KI实验的流动细胞学分析表明，接受AAV载体（图4）的细胞与RNP一起表示高达64%的EGFP阳性细胞（图5A），随后通过结点聚合酶链反应（PCR）（图5B）显示成功整合。细胞计数显示，所有样本在工程后3天内迅速恢复（图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细胞是从造血干细胞中提取的淋巴细胞，是适应性免疫系统幽默手臂的关键组成部分。他们之所以成为细胞疗法的候选者，是因为它们容易与周围血液隔离，体外扩张能力，以及体内寿命。此外，它们的正常生物功能——产生大量的抗体——可以用来表达大量的治疗性蛋白质，如抗感染的重组抗体，或用于治疗酶病变的酶。在这里，我们提供详细的方法，将原人类B细胞从外周血单核细胞（PBMCs）中分离，并在体外激活/扩大分离的B细胞。然后，我们演示了使用CRISPR/Cas9系统在B细胞中针对内源基因的部位特定KO所涉及的步骤。这种方法可以有效地利用各种基因的KO，用于研究感兴趣的基因的生物功能。然后，我们演示了使用CRISPR/Cas9系统以及重组的、与腺相关的病毒（rAAV）载体的步骤，以有效实现B细胞中转基因表达盒的现场特定集成。该协议共同提供了一个分步工程平台，可用于人类B细胞的初级研究基因的生物功能以及B细胞治疗的开发。

更新的扩展和激活协议使B细胞在7天内迅速膨胀到44倍（图1;n=3捐赠者）。在KO实验中，使用Trypan蓝色染色进行的B细胞计数显示，在24小时后的控制和CD19 KO样本中，超过80%的可行细胞的细胞恢复略有减少（图2A：p≥0.05，n = 3捐赠者）。这一结果表明，电波轻微影响整体B细胞健康。B细胞在输血后的第5天收集，用于流动细胞学和潮汐分析。控制和KO样本的?...

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

在这里，我们为CRISPR/Cas9基础基因组工程提供详细的分步协议，用于基因敲除（KO）和敲击（KI），以研究B细胞中基因的生物功能和B细胞治疗的开发。

使用CRISPR/Cas9的人类B细胞基因组工程

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 ...

This protocol allows researchers to precisely eliminate a gene in B cells to study the loss of function effects of that gene or to insert a trans gene for stable trans gene expression that can be used for research or therapeutic purposes. The main advantage of this method is that it provides a universal platform that makes it possible to easily and precisely engineer B cells with high efficiencies. This method can be used to engineer B cells to express recombinant antibodies or enzymes and then transfer those B cells to treat infections or in enzymopathies.To begin, thaw B cells in a 37 degree Celsius water bath. While waiting, transfer two milliliters of pre warmed FBS into a sterile, 15-milliliter conical tube. Once the B cells are completely thawed, immediately add one milliliter of pre-warmed FBS, drop-wise, into the sample.Incubate at room temperature for one minute. Gently pipette to resuspend the sample and transfer the whole volume, drop-wise, into a conical tube containing two milliliters of prewarmed FBS. Bring the volume to 15 milliliters with sterile PBS, then cap the tube and invert it gently two to three times.Centrifuge the sample at 400 times G for five minutes, then discard the supernatant without disturbing the cell pellet. Resuspend the pellet with one milliliter of pre-equilibrated B-cell expansion medium and count the cells. The total cell number should be approximately 10 to the seventh cells.Transfer the cells into a flask containing 20 milliliters of the pre-equilibrated B-cell expansion medium. The final concentration of the cells, should be approximately five times 10 to the fifth cells per milliliter. Incubate the flask vertically in a tissue culture incubator.Prepare the CRISPR/Cas9 transfecting substrate by mixing one microgram of chemically modified sgRNA with 1.5 micrograms of chemically modified streptococcus pyogenes Cas9 nucleus mRNA per transfection reaction. For control, add one microliter of TE buffer, instead of sgRNA, into a 0.2-milliliter tube of an eight tube strip. Turn on an electroporator and prepare the nuclear infection reagents.Count and transfer 1 million B cells per transfection reaction, into a sterile conical tube. Bring the volume to 15 milliliters with sterile PBS and centrifuge at 400 times G for five minutes. After centrifugation, discard the supernatant.Resuspend the cells with 10 milliliters of sterile PBS and centrifuge at 400 times G for five minutes, then discard the supernatant completely without disturbing the cell pellet. Add 0.5 micrograms of chemically modified GFP mRNA per 1 million B cells to the cell pellet. Resuspend the pellet with 20 microliters of primary cell transfection reagent per 1 million B cells and mix gently by pipetting five to six times.Transfer 20.5 microliters per transfection reaction into the 0.2-milliliter tubes containing CRISPR/Cas9 reagent. Pipette up and down once to mix and transfer the entire volume into a transfection cuvette. Cap and tap the cuvette on the bench gently to ensure that the liquid covers the bottom of the cuvette.Use the human primary B-cell protocol on the electroporator for transfection. Rest the electroporated cells in the cuvette at room temperature for 15 minutes. Then transfer 80 microliters of the pre-equilibrated B-cell expansion medium into the transfection reaction in the cuvette.Place the cuvette in the tissue culture incubator for 30 minutes. Gently pipe head a couple of 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 one milliliter of the B-cell expansion medium. The final concentration of the cells should be 1 million cells per milliliter.Performing a gene knock-in experiment, transfer recombinant adeno associated type-six viral vector at 500, 000 multiplicity of infection into the appropriate well containing electroporated cells. Place the plate in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide with humidity. In the knockout experiment, the B-cell count showed more than 80%viable cells with a slight reduction in cell recovery in both the control and the CD19 knockout samples at 24 hours post electroporation.B cells were collected on day five post-transfection for flow cytometry and tide analysis. Representative scatterplots of the control and knockout samples showed 14 and 95%CD19 negative cells respectively, demonstrating a significant reduction in CD19 expression in the knockout samples. Chromatograms of genomic sequencing showed double peaks in the CD19 knockout B cells, indicating insertions or deletions of nucleotides post-CRISPR/Cas9 mediated double-stranded breaks.Indel analysis of the chromatographs of the knockout samples showed a high percent of indel formation at the CD19 locus, which is consistent with percent CD19 protein loss detected by flow cytometry. B cells from the knock-in experiment were collected on day 12, post engineering. Scatterplots showed 64%of EGFP positive cells in the sample that received the recombinant adeno associated type-six viral vector, together with RNP, whereas none or minimal EGFP positivity was observed in the control and vector-only samples respectively.A junction PCR amplification, showed 1.5 kilobase pair amplicons in the knock-in sample and no PCR product was observed in either the control or vector-only sample. Cell counts showed that the engineering process affects cell recovery in the knock-in sample more than the control or the vector-only samples. However, all samples quickly rebounded within three days after engineering.This procedure can also be used to introduce base editor agents to alter single base pairs in order to induce or correct single point mutations in the B-cell genome. It could also be used to introduce premature stop codons or to alter splice sites, allowing for multiple gene knockouts without the risk of chromosomal translocations. Before this technique was developed, engineering human B cells would inquire the use of other more challenging gene engineering approaches.This technique is an easier, less expensive and quicker alternative. It could also potentially be used to synthesize cell therapy products for clinical use.

Research

JoVE Journal

Biology

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

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

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

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

科研

生物学

Generation of Human CD40-activated B cells

人CD40激活的B细胞的生成

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

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

天真成人干细胞的分离从初级人类成纤维细胞培养

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

Mouse Genome Engineering Using Designer Nucleases

小鼠基因组工程使用Designer核酸酶

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

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

原代培养小鼠肠嗜铬细胞的全细胞电生理

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

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

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

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

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

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

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

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

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 ...