我们感谢布赖恩. 西塞罗在编辑手稿方面的帮助。

健康捐赠者巴菲外套被获取了作为来源材料从中央俄亥俄区域美国红十字。本研究决定由全国儿童医院机构审查委员会进行豁免研究。
1. 人类 NK 细胞的纯化和扩展
<ol><li>隔离 PBMCs 从巴菲大衣12。<ol>
<li>35毫升的巴菲涂层样品在15毫升的 Ficoll-Paque。</li>
		<li>离心机在 400 x g 20 分钟没有刹车和收集 PBMC 从接口。</li>
		<li>通过增加至少一个同等体积的 PBS, 离心5分钟, 并吸去中国人民银行的洗涤, 清洗恢复的 PBMCs 三次。NK 细胞可以在这个阶段被 RosettesSep13隔离。</li>
	</ol>
</li>
	<li>在0、7和14天, 用辐照的 10 x 106 mbIL21-expressing 馈线细胞刺激 NK 细胞。用新鲜的 RPMI 替换介质, 每隔一天 IL-2 10% 只血清、1% 谷氨酰胺、1% 青霉素链霉素和100毫升/mL。</li>
</ol>2. gRNA 设计与选型
<ol><li>使用在线工具,例如, NCBI, 合奏, 选择特定的基因组定位点。 例子。 说明: 转化生长因子β受体 2 (TGFBRR2) 胞外区 查看记录: PF08917 查看 InterPro: IPR015013 位置:49-157 aa 目标序列: TGFBR2 基因外显子 4 (ENSG00000163513)</li>
	<li>要设计 gRNAs, 请使用 CRISPR 设计 web 工具, 如 http://crispr.mit.edu 和 "Benchling"。<ol>
<li>输入在步骤 2.1中选择的 DNA 序列。选择人 (hg 19) 作为目标基因组。CRISPR 参考线 (20 核苷酸后跟 PAM 序列: NGG) 将从先前输入的序列扫描。它还显示了在选定的基因组中可能存在的非目标匹配。</li>
		<li>根据目标和非目标利率选择最佳的三 gRNAs, 得分最高。例如,表 1显示了设计的 CRISPR rna, 目标为 TGFBR2 基因的外显子 4, CRISPR 设计 web 工具建议。</li>
	</ol>
</li>
	<li>命令 CRISPR rna 作为合成序列特定的 crRNAs。</li>
	<li>命令一个守恒的 transactivating RNA (tracrRNA) 通过与 crRNA 的部分同源性进行交互。</li>
</ol>3. 设计删除筛选底漆
<ol><li>设计引物跨越 gRNA 裂点的 T7E1 突变试验。</li>
	<li>使用引物至少 100 bp 离预测解裂点, 以确保小插入-删除 (indels) 在 sgRNA 的目标站点将出现在1.5% 琼脂糖凝胶后, 突变检测。表 2显示了用于放大 TGFBR2 胞外区的引物。</li>
</ol>4. 人类初级和扩大 NK 细胞的转导
注: 采用4D 系统进行电穿孔, 将 Cas9/RNPs 元素传导到 NK 中。
<ol><li>
单元准备
	<ol>
<li>
对于原 nk 细胞, 在 RPMI 培养基中孵化新鲜的 nk 细胞, 在 IL-2 100 毫升/mL 的情况下, 在5天进行电穿孔。每隔一天更换介质, 如前述和转导前一天。</li>
		<li>
对于扩张的 NK 细胞, 在0天用辐照的馈线细胞刺激细胞的比例为 1:1, 并在5或6或7天进行电穿孔。每隔一天更换介质, 如前述和转导前一天。</li>
		<li>在电穿孔的日子, 准备一个 T25 瓶填充8毫升新鲜 RPMI 包含100毫升/mL IL-2 的细胞进行电穿孔和预孵化烧瓶在湿润37°c 和 5% CO2孵化器。 注:经过2次或 3路刺激的解冻细胞或细胞在恢复后的任何时间都可以转。</li>
		<li>3-4 x 106细胞每条件为26µL 的转导组合。 注:电穿孔液中 NK 细胞浓度极高, 提高了转导率。</li>
		<li>用 PBS 冲洗细胞3次以去除所有的血清, 通常含有 RNase 活性。每次在 300 x g 处旋转8分钟。 注:考虑7电穿孔条件为 Cas/RNPs 作为单 gRNA (gRNA1, gRNA2, gRNA3) 和两个 gRNAs (gRNA1+gRNA2, gRNA1+gRNA3, gRNA2+gRNA3) 和一个控制, 没有 Cas9/RNPs 的组合。</li>
	</ol>
</li>
	<li>
形成 crRNA: tracerRNA/复合体
	<ol>
<li>并用重悬 crRNAs (gRNA1, gRNA2, gRNA3) 和 tracerRNA 1x TE 溶液的最终浓度为200微米。混合 2.2 ul 每200微米 gRNA 与200微米 tracerRNA, 如表 3所示。</li>
		<li>加热样品在95°c 5 分钟, 并允许冷却的长凳上到室温 (15-25 °c)。存储悬浮 rna 和 crRNA: tracerRNA/复杂在-20 °c 为以后使用。</li>
	</ol>
</li>
	<li>
形成 RNP 情结 注意:为了节省时间, 在洗涤步骤 4.1.5中形成 RNP 复合体。
	<ol>
<li>对于单 crRNA: tracrRNA 双工反应, 稀释 Cas9 限制性到36微米, 如表 4中的示例所示。</li>
		<li>crRNA 的组合转导: tracrRNA 复式, 稀释 Cas9 限制性到36微米, 如表 5中的例子所示。
</li>
		<li>添加 Cas9 限制性到 crRNA: tracrRNA 复式缓慢, 而旋转吸管提示, 三十年代至1分钟。</li>
		<li>在室温下孵育 15-20 分钟的混合物。如果不准备在孵化后使用混合物, 保持在冰上的混合物, 直到使用。</li>
	</ol>
</li>
	<li>
穿孔
	<ol>
<li>将整个补充剂添加到电穿孔溶液 P3, 并保持室温。</li>
		<li>并用重悬细胞颗粒 (3-4 x 106细胞从步骤 4.1.5) 在 20 ul P3 主要4D 电穿孔溶液。吹打时避免气泡。 注意: 细胞在 P3 溶液中不应该长时间留着。</li>
		<li>立即添加5µL RNP 复合物 (步骤 4.3) 的细胞悬浮。</li>
		<li>添加 1 ul 100 微米 Cas9 电穿孔增强剂的 Cas9/RNPs/cell 混合。</li>
		<li>转移 Cas9/RNPs/cell 混合成 20 ul 电穿孔带。</li>
		<li>轻轻地点击小条, 以确保样品覆盖底部的小条。</li>
		<li>启动4D 电穿孔系统, 选择EN-138程序。</li>
	</ol>
</li>
</ol>5. 后转导
<ol><li>让细胞在小条中休息3分钟。</li>
	<li>将预平衡培养基的80µL 添加到试管中, 并将样品轻轻地转移到烧瓶中。</li>
	<li>转导48小时后, 从 5 x 105细胞中提取基因组 DNA, 用于筛选。</li>
	<li>使用 Taq DNA 聚合酶试剂盒在步骤3.2 中设计的引物放大感兴趣的基因。</li>
	<li>形成 PCR 扩增子 heteroduplexes T7EI 消化和孵化的产品 30-60 分钟与 T7EI 酶37°c。 注:T7EI 检测是首选的筛选, 因为它是快速, 简单, 并提供了清洁电泳结果相比, 使用测量。但是, 此方法无法检测在 Cas9 RNPs 实验14中由非同源端连接 (NHEJ) 活动生成的 < 2 基的插入和删除。</li>
	<li>将1.5% 琼脂糖凝胶上的消化 DNA 在110伏上运行 30-45 分钟, 每15分钟可视化凝胶。</li>
	<li>用 mbIL21-expressing 馈线细胞刺激其余细胞的比例为1:1。</li>
	<li>五天后, 刺激提取 RNA 的基因表达水平使用 qPCR。</li>
	<li>按先前报告的12进行 calcein 化验。简单地, 用 calcein AM 加载目标细胞 (在显示的例子中, 使用了3µg/mL/100万 DAOY 细胞)。在 IL2 (100 毫升/ml) 加或减10的可溶性 TGFβ中过夜, 准备 NK 细胞进行细胞毒性测定。与 NK 细胞在一夜间休息时, 在相同的细胞因子中进行 calcein 化验。</li>
</ol>

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<li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+delivery+of+genome-editing+proteins+using+bioreducible+lipid+nanoparticles%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.</a> PNAS. 113 (11), 2868-2873 (2016).</li>
<li>Liang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+DNA-free+genome+editing+of+bread+wheat+using+CRISPR%2FCas9+ribonucleoprotein+complexes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.</a> Nature Communications. 8, 14261(2017).</li>
<li>DeWitt, M. A., Corn, J. E., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+editing+via+delivery+of+Cas9+ribonucleoprotein%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Genome editing via delivery of Cas9 ribonucleoprotein.</a> Methods. , 9-15 (2017).</li>
<li>Rezvani, K., Rouce, R., Liu, E., Shpall, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+Natural+Killer+Cells+for+Cancer+Immunotherapy%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Engineering Natural Killer Cells for Cancer Immunotherapy.</a> Molecular Therapy. 25 (8), 1769-1781 (2017).</li>
<li>Sutlu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells%3A+implications+for+gene+therapy%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Human Gene Therapy. 23 (10), 1090-1100 (2012).</li>
<li>Viel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta+inhibits+the+activation+and+functions+of+NK+cells+by+repressing+the+mTOR+pathway%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">TGF-beta inhibits the activation and functions of NK cells by repressing the mTOR pathway.</a> Science Signaling. 9 (415), (2016).</li>
<li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion%2C+purification%2C+and+functional+assessment+of+human+peripheral+blood+NK+cells%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> JoVE. (48), (2011).</li>
<li>Lee, D. A., Verneris, M. R., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acquisition%2C+preparation%2C+and+functional+assessment+of+human+NK+cells+for+adoptive+immunotherapy%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Acquisition, preparation, and functional assessment of human NK cells for adoptive immunotherapy.</a> Methods in Molecular Biology. , 61-77 (2010).</li>
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DAL 服务/已担任信使治疗, 黑曜石疗法, Intellia 疗法, 默克研究实验室和 Miltenyi Biotec 的顾问, 并在 CytoSen 疗法的公平/领导。

对 NK 细胞的 DNA 依赖性修饰已经挑战了4,9。因此, 我们直接介绍了一个综合预制 ribonucleoprotein (RNPs) 复合体和 Cas9 蛋白作为纯化蛋白成初级和扩大 NK 细胞8。这种方法使我们能够消除由 RNA 聚合酶 II 开始的封盖、尾矿和其他转录和平移过程, 这可能会导致 NK 细胞凋亡与 DNA 相关的转导方法有关。
此外, 这里报告的方法使用纯化的 Cas9 蛋白复合为 Cas9/RNPs, 这是立即激活后, 电穿孔和迅速退化, 从而增加目标和减少非目标影响的现行协议5,6,7,16. 此外, 优化一种新的电穿孔方法, 以高效率的传感器 Cas9/RNP 是另一个关键步骤, 这里介绍, 适用于任何其他基因的兴趣。这种方法可以扩大, 以修改更多的 NK 细胞使用商用更大的电穿孔小试管 (没有显示数据)。
总之, 利用上述方法, Cas9/RNPs 可用于基因修饰人类原发性和扩张 NK 细胞进行肿瘤免疫治疗。我们的研究结果还表明, 成功地剔除TGFBR2 胞外区基因导致这些改良的 NK 细胞成为 TGFβ1抵抗11。
结合 RNP 交付与模板 DNA 的来源 (如自然 recombinogenic 腺相关病毒 (AAV) 捐献者的载体) 可以通过同源重组3,18,19使特定地点的基因插入.

癌症免疫治疗近年来取得了进展。基因修饰的嵌合体抗原受体 (车载) T 细胞是成功地应用于癌症免疫治疗的工程免疫细胞的一个很好的例子。这些细胞最近被 FDA 批准用于治疗 CD19 + B 细胞恶性肿瘤, 但迄今为止成功仅限于携带少量多弹头抗原的疾病, 而针对这种有限的抗原曲目容易发生免疫逃逸的失败。此外, 由于异基因 t 细胞引起的移植物抗宿主病的风险, 汽车 t 细胞一直关注自体 t 细胞的使用。相反, NK 细胞能够以抗原无关的方式杀死肿瘤靶点, 不会引起 GvHD, 这使他们成为癌症免疫治疗6、7、8、9的好候选者。
CRISPR/Cas9 技术最近在工程免疫细胞中得到了应用, 但用质粒对 NK 细胞进行基因重组一直是很有挑战性的。这是由于在 DNA 依赖的情况下, 转基因分娩的困难, 如慢病毒载体和逆转录病毒转导导致大量程序相关的 nk 细胞凋亡和有限的基因工程 nk 细胞的生产4,9。
许多先天免疫细胞表达了与病原体相关的分子模式的高水平的受体, 如维甲酸诱导基因 i (钻机 i), 这使更多的识别外国 DNA。在使用基于 DNA 的基因修饰方法10时, 抑制这些通路使 NK 细胞的转导效率提高。
本文介绍了利用 Cas9 ribonucleoprotein 配合物 (Cas9/RNPs) 对原发性和扩张的人 NK 细胞进行无 DNA 基因组编辑的方法。Cas9/RNPs 由三组分组成, 重组 Cas9 蛋白与合成的单导 RNA 组成的复合 crRNA 和 tracerRNA。这些 Cas9/RNPs 能够分裂基因组目标, 与国外 DNA 依赖的方法相比, 由于其作为功能性复合物的交付。此外, 快速清除细胞中的 Cas9/RNPs 可减少诱导凋亡等非靶向效应。因此, 它们可以用来产生敲出, 或与 DNA 结合, 以同源重组6,7。结果表明, Cas9/RNPs 电穿孔是克服 NK 细胞遗传修饰的早期约束的一种简便、相对有效的方法。
TGFβ是抑制 NK 细胞活化和功能的主要免疫抑制因子。有人建议, 靶向 TGFβ通路可以增加免疫细胞功能。我们针对的区域编码 TGBR2 胞外区绑定 TGFβ11.结果表明, 该基因 mRNA 表达水平显著下降, 进一步证明改良后的 NK 细胞对 TGFβ有抵抗力。此外, 改良后的细胞保持生存能力和增殖潜能, 因为它们能够在电穿孔后使用辐照的馈线细胞扩大.因此, 下面的方法是一个有希望的方法, 基因操作 NK 细胞为任何进一步的临床或研究目的。

<table><tbody><tr><td>RosetteSep&amp;trade; Human NK Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15065</td><td>The RosetteSep&amp;trade; Human NK Cell Enrichment Cocktail is designed to isolate NK cells from whole blood by negative selection.</td></tr><tr><td>Ficoll-Paque&amp;reg; PLUS</td><td>GE Healthcare - Life Sciences</td><td>17-1440-02</td><td></td></tr><tr><td>Alt-R&amp;reg; CRISPR-Cas9 tracrRNA</td><td>Integrated DNA Technologies</td><td>1072532</td><td>TracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme</td></tr><tr><td>Alt-R&amp;reg; CRISPR-Cas9 crRNA</td><td>Integrated DNA Technologies</td><td></td><td>Synthetically produced as Alt-R&amp;reg; CRISPR-Cas9 crRNA, based on the sequence of designed gRNA targeting the gene of intrest</td></tr><tr><td>Alt-R&amp;reg; Genome Editing Detection Kit</td><td>Integrated DNA Technologies</td><td>1075931</td><td>Each kit contains T7EI endonuclease, T7EI reaction buffer, and T7EI assay controls.</td></tr><tr><td>Platinum&amp;reg; Taq DNA Polymerase High Fidelity</td><td>Invitrogen</td><td>11304-011</td><td></td></tr><tr><td>4D-Nucleofector&amp;trade; System</td><td>Lonza</td><td>AAF-1002B</td><td></td></tr><tr><td>Human recombinant IL-2 Protein</td><td>Novartis</td><td>65483-0116-07</td><td></td></tr><tr><td>P3 Primary Cell 4D-Nucleofector&amp;trade; X Kit</td><td>Lonza</td><td>V4XP-3032</td><td>Contains pmaxGFP&amp;trade; Vector, Nucleofector&amp;trade; Solution, Supplement, 16-well Nucleocuvette&amp;trade; Strips</td></tr><tr><td>Non-targeting: Custom siRNA, Standard 0.05 2mol ON-TARGETplus</td><td>Dharmacon</td><td>CTM-360019</td><td></td></tr><tr><td>Alt-R&amp;reg; S.p. Cas9 Nuclease 3NLS</td><td>Integrated DNA Technologies</td><td>1074181</td><td>Cas9 Nuclease</td></tr><tr><td>DNeasy&amp;reg; Blood &amp;amp; Tissue Handbook</td><td>Qiagen</td><td>69504</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Calcein AM</td><td>ThermoFisher</td><td>C3099</td><td></td></tr><tr><td>TGF&amp;beta;</td><td>Biolegend</td><td>580706</td><td></td></tr><tr><td>Alt-R&amp;reg; CRISPR-Cas9 Control Kit, Human</td><td>Integrated DNA Technologies</td><td>1072554</td><td>Includes tracrRNA, HPRT positive control crRNA, negative control crRNA#1, HPRT Primer Mix, and Nuclease-Free Duplex Buffer.</td></tr><tr><td>IDTE pH 7.5 (1X TE Solution)</td><td>Integrated DNA Technologies</td><td>11-01-02-02</td><td></td></tr><tr><td>Alt-R&amp;reg; Cas9 Electroporation Enhancer</td><td>Integrated DNA Technologies</td><td>1075915</td><td>Cas9 Electroporation Enhancer</td></tr></tbody></table>

generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins

CRISPR/Cas9 技术正在加速许多细胞类型的基因组工程, 但到目前为止, 基因传递和稳定的基因修饰在原 NK 细胞中具有挑战性。例如, 使用慢病毒载体或逆转录病毒转导的转基因分娩导致转基因 nk 细胞的产量有限, 这是由于大量程序相关的 nk 细胞凋亡所致。我们这里描述了一个无 DNA 的方法, 基因组编辑的人原发性和扩大 NK 细胞使用 Cas9 ribonucleoprotein 配合物 (Cas9/RNPs)。这种方法可以有效地剔除 NK 细胞中的 TGFBR2和HPRT1 基因。rt-pcr 数据显示, 基因表达水平显著下降, 一个代表性细胞产品的细胞毒性试验表明, RNP 修饰 NK 细胞对 TGFβ的敏感性较低。经辐照 mbIL21-expressing 馈线细胞刺激后, 基因修饰细胞可扩大电穿孔。

电穿孔效率
为了优化 Cas9/RNPs 的电穿孔, 我们对16种不同的方案进行了实验, 将 GFP 非靶向 siRNA 和 DNA 质粒转导到 NK 细胞。流式细胞仪检测表明, EN-138的细胞活力和转导效率的百分比最高 (35% 活 GFP 阳性细胞) 为两个粒子 (图 1 &图 2)。有趣的是, 使用这个程序 Cas9/RNPs 电穿孔的效率较高, 因为我们看到了60% 降低 TGFBR2 mRNA 表达水平 (图 5)。此外, 转基因 NK 细胞可以生长和扩展30天, 并保存 (未显示数据)。
突变检测
Cas9/RNPs 含有 gRNA2, gRNA1+gRNA2 和 gRNA3 成功 TGFBR2 胞外区基因敲除, 但 gRNA1 单独没有作出任何 T7E1 可检测 indels (图 3)。此外,图 4表明使用商业提供的 gRNAs 成功地挖空了人类 HPRT1 (嘌呤 phosphoribosyltransferase 1)在扩张的人类 NK 细胞中。根据波段密度测定, 使用 gRNA1+gRNA2 的比例 indel 率导致 TGFBR2 改良 nk 细胞的34% 波段, gRNA3 81%, HPRT 基因修饰 nk 细胞25%。
基因表达水平测定
作为我们结果的代表,图 5显示了 Cas9/RNPs (gRNA1+gRNA2) 对 TGFBR2 胞外区 mRNA 生产水平的影响, 通过 rt-pcr 分析。如图所示, 靶向基因的 mRNA 表达水平明显降低。
细胞 毒性
如图 6所示, 在孵化 gRNA1+gRNA2 后, gRNA2 和 gRNA3 Cas9/RNPs 修饰细胞与 TGFβ1, 共培养 DAOY 细胞;经改良的细胞与对照组相比, 其细胞毒性水平没有明显降低, IL-2 在夜间传播。结果表明, Cas9/RNPs 修饰细胞在 TGFβ1存在时保留其细胞毒性作用, 表明修饰细胞 TGFβ1抗性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58237/58237fig1.jpg"> 图 1.这些数字显示了 siRNA 和质粒 DNA 表达 GFP 在 NK 细胞中的电穿孔效率使用 EN-138 程序。如下所示, NK 细胞的生存能力为 77.5%, 35% 的活细胞为 GFP 阳性。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58237/58237fig2.jpg"> 图 2.这个数字显示了另一个16程序(DN-100)测试的电穿孔优化的可行性和效率。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58237/58237fig3.jpg"> 图 3.Cas9/RNPs-mediated TGFBR2 敲除在花费(a)主要 NK 细胞(b)用 T7E1 突变测定法测量。T7E1 酶识别和裂解不匹配的 DNA。每个小波段 (蓝色箭头) 代表被消化的脱氧核糖核酸片断运载 indel。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58237/58237fig4.jpg"> 图 4.Cas9/RNPs 介导的 HPRT 在扩张的 NK 细胞中 T7E1 突变测定的干扰。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58237/58237fig5.jpg"> 图 5.应用 rt-pcr 方法 Cas9/RNPs (gRNA1+gRNA2) 介绍 CRISPR 改良 NK 细胞中 TGFBR2 胞外区的 mRNA 表达水平。GAPDH 被用作内源性控制基因。RNA 水平的降低表明 TGFBR2 基因的中断 (平均的扫描电镜, P 值 < 0.0001)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58237/58237fig6.jpg"> 图 6.使用具有代表性的 Cas9/RNPs 修饰 (gRNA1+gRNA2、gRNA2 和 gRNA3) NK 细胞的细胞毒性试验表明, TGFβ1细胞的夜间孵化并没有显著降低其溶解 DAOY 细胞的能力。b.与非改良 nk 细胞相比, Cas9/RNP 修饰 nk 细胞 (gRNA2 和 gRNA3) 对 TGFβ1的敏感性较小 (均为 SEM)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58237/58237fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>gRNA 不。</td>
			<td colspan="2">gRNA 序列</td>
			<td colspan="5">订购为合成 crRNA</td>
		</tr>
<tr>
<td>gRNA1</td>
			<td colspan="2">5 CCCCTACCATGACTTTATTC 3</td>
			<td colspan="5">/AltR1/rArGrUrCrArUrGrGrUrArGrGrGrGrArGrCrUrUrGrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AltR2/</td>
		</tr>
<tr>
<td>gRNA2</td>
			<td colspan="2">5 ATTGCACTCATCAGAGCTAC 3</td>
			<td colspan="5">/AltR1/rArUrUrGrCrArCrUrCrArUrCrArGrArGrCrUrArCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AltR2/</td>
		</tr>
<tr>
<td>gRNA3</td>
			<td colspan="2">5 AGTCATGGTAGGGGAGCTTG 3</td>
			<td colspan="5">/AltR1/rArG rUrCrA rUrGrG rArGrC rUrArGrGrGrG rUrUrG rGrUrUrUrUrA rGrArG rCrUrA rUrGrCrU/AltR2/</td>
		</tr>
</tbody></table>表1。三设计 gRNAs 靶向外显子 4 TGFBR2 胞外区为合成 crRNA。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>TGFBR 2 胞外区底漆</td>
			<td>5 TGC AGG TGA TTA T3</td>
		</tr>
<tr>
<td>TGFBR2 胞外区底漆转速</td>
			<td>5 GGG CCT TTA 3</td>
		</tr>
</tbody></table>表2。用于放大 TGFBR2 胞外区基因的引物
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>金额 (uL)</td>
		</tr>
<tr>
<td>200µM crRNA</td>
			<td>2。2</td>
		</tr>
<tr>
<td>200µM 示踪 RNA</td>
			<td>2。2</td>
		</tr>
<tr>
<td>IDTE 缓冲区</td>
			<td>5。6</td>
		</tr>
<tr>
<td>最终产品</td>
			<td>10</td>
		</tr>
</tbody></table>表3。形成 crRNA: tracerRNA/复用200µM rna
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>金额 (µL)</td>
		</tr>
<tr>
<td>Pbs</td>
			<td>1</td>
		</tr>
<tr>
<td>crRNA: tracrRNA 双工 (从步骤 4.2)</td>
			<td>2 (200 pmol)</td>
		</tr>
<tr>
<td>Alt R Cas9 限制性 (61 µM 库存)</td>
			<td>2</td>
		</tr>
<tr>
<td>总容积</td>
			<td>5 ul</td>
		</tr>
</tbody></table>表4。单 crRNA: tracrRNA 双面反应, 稀释 Cas9 限制性至36µM。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>金额 (µL)</td>
		</tr>
<tr>
<td>Pbs</td>
			<td>1</td>
		</tr>
<tr>
<td>crRNA: tracrRNA 双工 (gRNA1)</td>
			<td>1 (100 pmol)</td>
		</tr>
<tr>
<td>crRNA: tracrRNA 双工 (gRNA2)</td>
			<td>1 (100 pmol)</td>
		</tr>
<tr>
<td>Alt R Cas9 限制性</td>
			<td>2</td>
		</tr>
<tr>
<td>总容积</td>
			<td>5µL</td>
		</tr>
</tbody></table>表5。crRNA 的组合转导: tracrRNA 复式稀释 Cas9 限制性至36µM。

Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

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

在这里, 我们提出了一个协议, 基因改造主要或扩大人类自然杀手 (NK) 细胞使用Cas9 Ribonucleoproteins (Cas9/RNPs)。通过使用该协议, 我们生成的人 NK 细胞缺乏转化生长 factor–b 受体 2 (TGFBR2) 和嘌呤 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 ...

generation-knock-out-primary-expanded-human-nk-cells-using-cas9

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  Nationwide Children's Hospital. 在这里, 我们提出了一个协议, 基因改造主要或扩大人类自然杀手 (NK) 细胞使用Cas9 Ribonucleoproteins (Cas9/RNPs)。通过使用该协议, 我们生成的人 NK 细胞缺乏转化生长 factor–b 受体 2 (TGFBR2) 和嘌呤 phosphoribosyltransferase 1 (HPRT1)。

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

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

遗传学

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. ...

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

使用慢病毒介导的CRISPR / Cas9基因编辑开发敲除肌肉细胞系

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

免疫与感染

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

THP-1 细胞中基于电穿孔的 CRISPR-Cas9 介导的基因敲除和单细胞克隆分离

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

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.

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.

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 ...

生物学

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

通过病毒样颗粒（"纳米刀片"）在永生化和原代细胞中递送Cas9 / sgRNA核糖核蛋白复合物

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System

This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated guide RNA plasmids and lentiviral packaging plasmids to achieve RIP1 gene knockout. The protocol addresses challenges and improvements to traditional CRISPR methods, enabling its replication for future cell death studies. The resulting mutant cells can be used to investigate mechanistic changes in cell death, where functional RIP1 proteins would otherwise play a role. Viability assays demonstrated a significant reduction in cell death in knockout cells following necroptosis induction. Fluorescence microscopy revealed a marked decrease in mitochondrial reactive oxygen species (ROS) in knockout cells under the same conditions. Together, these functional assays confirm the loss of RIP1 protein. Optimized for use with U937 human monocytes, this procedure may also be adapted to target other key cell death regulators, yielding functional, non-lethal mutants. Potential pitfalls are addressed throughout to provide insights into challenges that may arise during mutant generation.

As CRISPR-related protocols become increasingly useful and accessible, complications and obstacles can still arise under specific experimental conditions. This protocol outlines the creation of a receptor-interacting serine/threonine-protein kinase 1 (RIPK1/RIP1) knockout human cell line using CRISPR/Cas9 and highlights potential challenges encountered during this process.

使用 CRISPR-Cas9 系统生成 RIP1 敲除 U937 细胞系

This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated ...

A CRISPR-Cas9 Technique for Gene Editing in T Cells

Source: Agarwal, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62299">Production of Human CRISPR-Engineered CAR-T Cells.</a> J. Vis. Exp. (2021).
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell&#39;s non-homologous end-joining mechanism, leading to gene knockout.

用于 T 细胞基因编辑的 CRISPR-Cas9 技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Designing of sgRNAs and gene ...