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 寡核苷酸重组为浓度为 1 毫克/mL。</li>
	<li>将 CD40 配体 （CD40L） 重组为浓度为 100μg/mL。</li>
	<li>重组重组人类IL-10（rhIL-10），浓度为50微克/mL。</li>
	<li>重组重组人类IL-15（rhIL-15），浓度为10微克/mL。 注意:将每种补充剂保持在-20°C至-80°C的小参考中长达6个月。</li>
</ol>2. 准备基础介质
<ol><li>结合B细胞基底介质与5%（v/v）介质补充体外免疫细胞扩张（如CTS免疫细胞SR）和1%（v/v）青霉素和链霉素。</li>
	<li>使用 0.22μm 滤芯适配器将基底介质过滤消毒到消毒瓶中。</li>
	<li>将基底介质保持在 4 °C，最长达 1 个月。</li>
</ol>3. 准备B细胞扩展介质
<ol><li>将基底介质所需的量转移到无菌容器中，以 5 × 105 细胞/mL 培养 B 细胞。</li>
	<li>以 1 μg/mL CpG、100 ng/mL CD40L、50 ng/mL rhIL-10 和 10 ng/mL rhIL-15 补充基础介质。</li>
	<li>使用 0.22 μm 过滤器过滤 B 细胞扩展介质。</li>
	<li>在 37 °C、5% CO2、 湿度至少 30 分钟之前，在组织培养孵化器中平衡 B 细胞膨胀介质。 注意:准备新鲜的B细胞扩展介质，使用一天。 不要 准备 B 细胞扩展介质使用多天。这种介质配方鼓励记忆B细胞的增殖，并激活原发性人类B细胞。</li>
</ol>4. 人类B细胞净化和扩张
注:按照制造商的指示，在温度控制的冷冻容器中加入 99–100% 异丙醇，并在开始第 4.1 步之前将冷冻容器冷却在 4 °C。
<ol><li>从白血病样本中分离 PBMC
	<ol>
<li>将白血病样本（约 8–10 mL）转移到无菌的 50 mL 锥形管中。</li>
		<li>将体积调高至 35 mL，无菌 1x 磷酸盐缓冲盐水 （PBS）。</li>
		<li>在 15 mL 密度梯度介质上仔细分层 35 mL leuka 磷化样本。</li>
		<li>离心机在500× 克 25分钟没有刹车，去除等离子层，而不会干扰抛光外套（PBMC层），从接口收集PCBCs，并转移到一个新的无菌50mL圆锥管。</li>
		<li>将 PBMC 与 1 倍 PBS 一起提起 50 mL。</li>
		<li>离心机在500× 克 5分钟没有刹车。在不干扰 PBMC 颗粒的情况下取出超自然，PBMC 颗粒可能呈红色。</li>
		<li>加入7mL的铵-氯化钾-溶解缓冲器，移液器3次混合良好，并在室温下（RT）孵育3分钟。</li>
		<li>将音量调高至 50 mL，并配备 1 倍 PBS。</li>
		<li>离心机在400× 克 5分钟低阻力制动器。在不干扰颗粒的情况下取出超自然。弹丸应该看起来粉红色或白色。 注意:要继续培养刚分离的 B 细胞，请在开始 B 细胞分离之前准备 B 细胞扩展介质（步骤 4.2）。</li>
	</ol></li>
	<li>使用人类初级 B 细胞负隔离套件从 PBMC 中分离 B 细胞
	<ol>
<li>在隔离缓冲区中将 PBMC 恢复为浓度为 5 × 107 个单元/mL。 注意:如果 PBMC 总数小于 5 × 107 个单元，请缩小隔离缓冲器的体积，以保持 5 × 107 个单元格/mL。细胞悬架的最小和最大体积分别为 0.25 mL 和 8 mL。</li>
		<li>将高达 8 mL（5 × 107 个电池/mL）转移到带盖子的无菌聚丙烯圆底管中。</li>
		<li>在 PBMC 中添加 50μL/mL 的鸡尾酒增强剂。</li>
		<li>在 PBMC 中加入 50μL/mL 的隔离鸡尾酒，盖上管子，然后倒置 2~3 次混合。</li>
		<li>在 RT 孵化 5 分钟;在孵化的第4 分钟，涡流磁性微珠至少30秒。</li>
		<li>每 1 mL PBMC 传输 50μL 的磁性微珠，封盖管子，并倒置 2–3 次混合。</li>
		<li>使用隔离缓冲区充值至 10 mL，并轻轻上下 2–3 次移液器。</li>
		<li>将管子放在磁站中，在 RT 孵化 3 分钟。</li>
		<li>将磁铁和管子保持在一起，在一个动作中，将磁铁和管子倒在一起，将细胞悬架倒入新管中。丢弃旧管子。</li>
		<li>重复步骤 4.2.8（将孵化时间缩短到 2 分钟），并将 B 细胞悬架倒入干净的圆锥形管中。</li>
		<li>富集的 B 细胞已准备好使用。如果立即使用细胞，请继续使用第 4.3 节（人类 B 细胞扩展）。通过流动细胞学（可选）检查分离 B 细胞的纯度。如果细胞在使用前要冻结，请继续踩踏 4.2.12。</li>
		<li>要冻结B细胞，离心机在400× 克 5分钟，并丢弃超自然分子，而不会干扰颗粒。</li>
		<li>将细胞在冷冻介质中以107 个细胞/mL进行恢复，并引用1mL/低温。</li>
		<li>将冷冻容器放在冰冷的冷冻容器中，并在 -80 °C 过夜;然后，将冷冻低温转移到液氮罐中:保持冷冻细胞长达1年。 注:分离B细胞的预期产量占全氟辛起伏碳化合物总量的2%-8%，生存能力为95%-99%。</li>
	</ol></li>
	<li>人类B细胞扩张 注意:如果使用刚分离的 B 细胞，请跳过步骤 4.3.1– 4.3.5。数数细胞并将所需细胞数转移到无菌圆锥管中，并继续执行步骤 4.3.6。<ol>
<li>在解冻B细胞之前，在水浴中预热胎儿牛血清（FBS）。准备20mL的B细胞膨胀介质，转移到T25烧瓶，并在组织培养孵化器中预平衡介质（在37°C，5%CO2，湿度），至少15分钟使用前。</li>
		<li>在37°C的水浴中解冻B细胞。在等待时，将 2 毫升预热 FBS 转移到无菌的 15 mL 圆锥管中。</li>
		<li>B细胞完全解冻后，立即在样品中加入1毫升预热的FBS。在 RT 孵化 1 分钟。</li>
		<li>轻轻移液器，以补充样品，并转移整个体积，下降，到一个圆锥管含有2毫升预热的FBS。</li>
		<li>将体积调高至 15 mL，带无菌 1 倍 PBS、盖，并轻轻地将管子倒置 2–3 倍。</li>
		<li>离心机在 400 × g 5 分钟。</li>
		<li>在不干扰细胞颗粒的情况下丢弃超细剂，用1mL的预平衡B细胞膨胀介质补充细胞颗粒，并计算细胞数。总细胞数应约为107 个单元格。</li>
		<li>将细胞转移到含有 20 mL 预平衡 B 细胞膨胀介质的烧瓶中。细胞的最终浓度应约为5×105 细胞/mL。</li>
		<li>将烧瓶垂直孵化在组织培养孵化器中。</li>
		<li>通过将 B 细胞的整个体积转移到无菌圆锥管中，并重复步骤 4.3.6–4.3.9，每 2 天完全刷新一次扩展介质。 注:T25烧瓶可容纳10-20 mL的介质:T75 烧瓶可容纳高达 20–60 mL 的介质。</li>
	</ol></li>
</ol>5. 初级人类B细胞工程
<ol><li>工程师 B 细胞在扩展/激活后以 48 ± 2 h 的速度工作，以获得最佳效果。准备B细胞膨胀介质，将介质的1mL放入48井组织培养板中，并在使用前至少15分钟在组织培养孵化器中预平衡。 注意:在为感兴趣的基因设计CRISPR/Cas9 sgRNA时，请按照下面概述的步骤操作。 - 使用在线工具11设计 sgRNA 。 - 在蛋白质的所有同位素中常见的外体上设计sgRNA。 - 从信誉良好的公司订购化学改装的 sgRNA。 -sgRNA通常以嗜血的形式出现:在无菌 DNase/无鼻塞三叶草 EDTA （TE） 缓冲区中重组 sgRNA，浓度为 1 μg/μL。</li>
	<li>通过将化学改性 sgRNA 的 1 μL （1μg/μL） 与化学改性链 球菌 Cas 9 （S.p. Cas9） 核糖核酸混合，准备 CRISPR/Cas9 变种基板。要进行控制，请使用 1 μL 的 TE 缓冲区，而不是 sgRNA。 注:当 CD19 sgRNA 用于基因 KO 实验时，请参阅 图 2 了解结果。当 AAVS1 sgRNA 用于基因 KI 实验时，请参阅 图 5 了解结果。<ul>
<li>将所有组件保留在冰上。</li>
	</ul></li>
	<li>轻轻混合，将每个反应的 2.5μL CRISPR/Cas9 变速基板传输到 0.2 mL 8 管条的管中;放在RT。</li>
	<li>打开核燃机（电极器），并准备 如表1所示的转染试剂。 注意:如果必要的话，把所有试剂都放在冰上，这是暂停的好步骤。准备恢复实验时，从冰中取出所有试剂。当使用 S.p. Cas9 蛋白质时，研究者 必须 通过将 1μg sgRNA 与 5 μg S.p. Cas9 蛋白质混合，避免任何气泡，并在 RT 中孵育混合物至少 20 分钟，然后再使用以获得最佳效果。</li>
	<li>计数并转移10个6 B细胞，每个转染反应到无菌圆锥管。</li>
	<li>将体积调高至 15 mL，无菌 1 倍 PBS，离心机在 400 × g 下 5 分钟。在等待时，准备主细胞转染试剂（表2），并放在RT。</li>
	<li>丢弃超自然的，而不会干扰细胞颗粒。</li>
	<li>用 10 mL 的无菌 1 倍 PBS 和离心机在 400 × g 下重新注入细胞颗粒 5 分钟。</li>
	<li>完全丢弃超纳特，而不会干扰细胞颗粒。</li>
	<li>每106 B细胞将0.5微克化学改性GFP mRNA（作为传染报告器）转移到细胞颗粒（可选）。</li>
	<li>每10个6B 细胞用20μL原细胞传染试剂补充细胞颗粒;通过管道轻轻混合5-6次。将 20.5 μL 的每次转染反应转移到包含 CRISPR/Cas9 转染基板 2.5 μL 的 8 管条的 0.2 mL 管中。</li>
	<li>向上和向下一 次 将整个音量 （23 μL） 混合并传输到传输库维特。轻轻盖住并轻敲长凳上的绒布，以确保液体覆盖库维特的底部。</li>
	<li>在核细胞核上使用人类原生B细胞协议进行转染。 注:核效应器（电容器）可放置在组织培养罩外使用。盖上小面包，确保不育。</li>
	<li>将电波化细胞在 RT 的库维特中休息 15 分钟。</li>
	<li>将预平衡 B 细胞膨胀介质的 80 μL 从组织培养板转移到库维特的转染反应中。将小面包放在组织培养孵化器中30分钟。</li>
	<li>轻轻移液几次，将样品的整个体积从库维特混合并转移到含有 1 mL B 细胞膨胀介质的 48 井组织培养板的适当井中。细胞的最终浓度应该是106 个细胞/mL。</li>
	<li>如果进行基因 KI 实验， 将 rAAV6 向量为 500，000 多种感染的载体转移到含有电波酸细胞的适当井中（大约 45 分钟后电波）。请参阅 图 4A中的示例 rAAV6 矢量构造。 注:例如:在没有CRISPR/Cas9或rAAV6矢量的情况下，对照样品将被电波解。仅矢量样品将电波化，无需 CRISPR/Cas9，然后与 rAAV6 矢量一起转导。KI 样品将用 CRISPR/Cas9 进行电波化，然后用 rAAV6 矢量进行转导。rAAV6 必须包含 HDR 目标 DSB 站点的上下同源臂。</li>
	<li>将盘子放在湿度为 37 °C 和 5% CO2 的组织培养孵化器中。</li>
	<li>计算细胞并记录工程后第 1 天的可行性。</li>
	<li>通过计算细胞，然后将整个细胞体积转移到一个干净的 1.5 mL 微中微富格管中，每 2 天刷新一次 B 细胞扩展介质。离心机在 400 × g 5 分钟， 并丢弃超母体， 而不会干扰颗粒。用100μL的新鲜B细胞膨胀介质补充细胞，并转移到24井组织培养板的井中。提起中等体积，在 5 × 105 细胞/mL 时实现最终细胞浓度。 注:48井板可容纳1mL中/井:一个24井板可以容纳高达2 mL中/井:12井板可容纳4mL中/井，6井板可容纳8mL中/井。</li>
	<li>允许工程细胞在进行下游分析（如流细胞学分析、TIDE 分析和交界 PCR）之前至少膨胀 5 天。</li>
</ol>

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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>

以M.J.J、K.L.和B..M S.为发明人，对制造和使用基因组编辑的B细胞的方法提出了专利。B.S..M是伊姆穆索夫特公司的顾问，并拥有该公司的股票。伊姆穆索夫特公司赞助了B.S..M实验室的研究。

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&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样本的代?...

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 ...

genome-engineering-of-primary-human-b-cells-using-crisprcas9

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.1K 次观看.  University of Minnesota. 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。

视频: 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.

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

科研

癌症研究

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.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

遗传学

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

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.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

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

原代骨髓来源的巨噬细胞中精确基因破坏的指南

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.

作者聚焦：在骨髓来源的巨噬细胞中进行高效的 CRISPR/Cas9 基因组编辑以实现精确的基因破坏

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.

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

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.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

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).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

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

免疫与感染

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.

使用CRISPR/Cas9的人类B细胞基因组工程

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利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

生产人类CRISPR工程的CAR-T细胞