这项工作由美国国立卫生研究院资助5R01AI144149资助。示意图是使用 BioRender 创建的。

1. sgRNA设计
注意：此步骤描述了靶序列的选择和sgRNA的设计。设计位于第一个大编码外显子中的指南是有帮助的，这样任何翻译的蛋白质在开放阅读框的早期就会被破坏。选择位于同一外显子内的靶序列也很有帮助，因为这将简化编辑效率的分析（步骤6）。该协议提供的基因组编辑示例使用靶向 Src 基因和 Cblb 基因的第一个外显子以及小鼠基因组的非编码 Rosa26 位点的sgRNA。
<ol><li>使用几种免费的在线设计工具之一鉴定 20 个要靶向的核苷酸基因组序列（表 1）。选择每个基因内不重叠的四到五个向导，因为 Cas9 活性因所选的特定向导而异，并且无法 先 验预测高活性向导。 注意：确保指南相对于目标位点序列的正确方向至关重要。设计工具通常以 5' 到 3' 的方向提供引导序列，而不管靶向哪条染色体链。要验证链方向，请检查靶向基因组序列的 3' 处是否存在 化脓性链球 菌 Cas9 （nGG） 的原间隔相邻基序 （PAM） 序列。sgRNA 序列不包括 PAM。</li>
</ol>2. sgRNA合成
注意：此步骤描述了如何使用PCR合成sgRNA以生成 用于体外 转录（IVT）的模板，然后使用离心柱纯化sgRNA（图1A）。定制合成 sgRNA 可通过多家供应商获得，作为 PCR/IVT 的替代品。
<ol><li>进行PCR生成T7 RNA聚合酶的IVT模板。PCR反应使用含有T7 RNA聚合酶启动子和反式激活CRISPR RNA（tracrRNA）序列的通用引物（表2），以及具有基因特异性引导序列的短单个引物。<ol><li>这是非扩增重叠延伸步骤。将PCR缓冲液稀释至1x，并再加入1mM MgCl2。然后，加入 0.25 μL 高保真 DNA 聚合酶、0.5 mM 脱氧核苷酸三磷酸 （dNTP）、0.02 mM 向导特异性引物和 0.02 mM T7 反向长通用引物（表 2），总体积为 46 μL。然后，将试管放入热循环仪中并运行两个循环：95°C15秒，37°C15秒和72°C15秒。在冰上冷却反应。</li>
		<li>这是PCR扩增步骤。向每个反应中加入 2 μL 25 mM 正向扩增引物和 2 μL 25 mM 反向扩增引物。最终反应体积为50μL，在95°C下变性30秒。 然后，运行 35 个循环：95 °C 15 秒，65 °C 20 秒和 72 °C 15 秒。在72°C下进行额外的延伸2分钟。</li>
		<li>在2%琼脂糖凝胶上检查PCR反应产物。重叠延伸PCR产生127 bp的dsDNA分子，其中T7启动子位于基因特异性20核苷酸向导的上游，tracrRNA紧邻下游（图1B）。</li>
	</ol></li>
	<li>IVT模板纯化：有许多方案和试剂盒可以很好地用于此。该协议使用固相可逆固定化（SPRI）珠。将 50 μL PCR 产物与 90 μL 微珠混合，并按照制造商的说明进行操作。通过加入 40 μL 缓冲液（5 mM Tris、0.1 mM 乙二胺四乙酸 [EDTA]）并在 65 °C 下加热 5 分钟来洗脱 DNA。</li>
	<li>使用260nm波长的吸收测量IVT模板的DNA浓度。IVT 需要至少 50 ng/μL 的 DNA 浓度。IVT模板可以储存在-20°C。</li>
	<li>IVT反应<ol><li>使用旨在产生高产量 IVT 的商业试剂盒（参见 材料表）。按照制造商的建议进行 IVT 反应。在20μL反应中使用250ng IVT模板，在37°C孵育4-16小时。</li>
		<li>通过在 2% 琼脂糖凝胶上运行 1 μL 反应来检查 IVT sgRNA 产物。应观察到明亮的RNA条带，其运行方式与70 bp dsDNA相似（图1C）。虽然不是强制性的，但为了获得更清晰和更分离的条带，请使用含有尿素的RNA样品缓冲液，并在上样前在65°C下使样品变性5分钟。 注意：由于RNA二级结构的差异，某些向导RNA可能会看到微弱的高分子量带和轻微的迁移差异。</li>
	</ol></li>
	<li>使 sgRNA IVT 产物去磷酸化，以最大限度地减少 RIG-I（一种外来 RNA 传感器）的激活和电穿孔后随后的细胞死亡。对于每 20 μL IVT 反应，在 1x CIP 缓冲液中加入 69 μL 分子级水和 15 U 犊牛肠磷酸酶 （CIP）。在37°C孵育2小时。</li>
	<li>降解 IVT 模板以最大限度地减少细胞 DNA 传感器的激活，细胞 DNA 传感器在电穿孔后触发细胞死亡。在每个 IVT 反应中，加入 2 U 不含 RNase 的 DNase。在37°C孵育15分钟。 IVT产品可以在-20°C下储存1天或-80°C储存数月。</li>
	<li>使用离心柱纯化IVT产品。对于此步骤，SPRI微球纯化试剂盒较差。<ol><li>通过向 IVT 产物中加入 220 μL 结合缓冲液，然后加入 1 mL 100% 分子生物学级乙醇，将 RNA 结合到色谱柱上。搅拌均匀并加载色谱柱。此后，请按照制造商的说明进行操作。在层流生物安全柜中进行最后的洗涤，以避免sgRNA的污染。</li>
		<li>在层流罩中进行洗脱以避免污染。使用 30 μL 无菌 10 mM Tris 缓冲液 （pH 8.0） 洗脱 RNA。</li>
	</ol></li>
	<li>通过测量波长为260nm的吸光度来测量sgRNA的浓度。 注：典型的sgRNA产量至少为100μg。</li>
	<li>将sgRNA储存在-80°C。 制作等分试样以避免超过三个冻融循环。</li>
</ol>3.电穿孔的准备
注意： 所有步骤都应在层流罩中执行，以避免污染。该方案使用具有10μL吸头的市售电穿孔系统（参见 材料表）。
<ol><li>使用前解冻BMDM48-72小时，并在非组织培养（TC）处理的平板上展开。 注意：每个反应需要 4 x 105 个细胞。</li>
	<li>对PCR管进行灭菌以进行反应组装。每个反应准备一个PCR管，并在热循环仪中在98°C下加热10分钟。 分批准备试管并密闭存放。准备使用时，将灭菌的PCR管放在冰上。</li>
	<li>用70%乙醇清洁移液器工作站，并将其放入层流罩中。将参数设置为 1,900 V、20 ms 和 1 个脉冲。</li>
	<li>小心地从包装中取出转染管以保持无菌状态，并用 3 mL “E” 缓冲液填充。在电穿孔之前，确保E缓冲液处于室温。将管子插入工作站并将其向下推，直到发出咔嗒声。</li>
	<li>对于每个反应，分装 2.5 μL 浓度为 1,100 ng/μL 的 sgRNA。 用 0.9 mM CaCl2 和 0.5 mM MgCl2 （PBS + Ca/Mg） 在冷磷酸盐缓冲盐水 （PBS） 中稀释 sgRNA。把它放在冰上。</li>
	<li>准备两个板：一个用于细胞的 12 孔未包被板和一个额外的 24 孔板用于容纳培养基。每次反应将 1.5 mL 无抗生素培养基等分到 24 孔板的孔中。</li>
	<li>制备Cas9蛋白。无菌纯化的 Cas9 蛋白有几家商业和学术供应商。用冷PBS + Ca / Mg稀释Cas9至终浓度为20μM。对于每个电穿孔反应，将 1 μL 20 μM Cas9 分装到无菌 PCR 管中。放在冰上。</li>
	<li>准备用于电穿孔的细胞。<ol><li>取出生长培养基。使用不含 CaCl2 和 MgCl2 （PBS-Ca/Mg） 的 PBS 洗涤细胞，以去除促进粘附的血清和二价阳离子。然后，加入PBS + 1mM EDTA并在室温下孵育约5分钟，直到细胞开始分离。</li>
		<li>轻轻地上下移液以分离细胞。将细胞转移到低蛋白结合的 15 mL 管中。将细胞以500× g 沉淀5分钟。 注意：巨噬细胞粘附在塑料器皿上。使用低蛋白结合离心管可显著提高细胞产量。</li>
		<li>快速工作以避免细胞在 PBS + EDTA 中长时间孵育。除去大部分上清液，在试管中留下约 1 mL 的上清液。将细胞重悬于剩余的上清液中，然后转移到低蛋白结合的1.5ml微量离心管中。</li>
		<li>取出一小等分的细胞进行计数。通过在室温下以500× g 离心5分钟来沉淀剩余的细胞。</li>
		<li>在离心过程中，计数细胞并将Cas9和sgRNA管加热至室温。</li>
		<li>从细胞沉淀中除去所有上清液。将细胞重悬于PBS + Ca/Mg中，浓度为4×107 个细胞/ mL（每10μL反应4×105 个细胞）。 注意：每个试剂盒都提供有限量的电穿孔缓冲液（缓冲液R，缓冲液T）。然而，我们发现使用PBS + Ca/Mg的电穿孔效率与专有缓冲液相当。</li>
		<li>在电穿孔不同基因的 sgRNA 时，将细胞分装到每个基因的不同低蛋白结合管中，以避免 sgRNA 之间的交叉污染。将细胞保持在室温下，直到准备好进行电穿孔。</li>
	</ol></li>
</ol>4. RNP组装
<ol><li>将sgRNA和Cas9保持在室温下，以避免沉淀。在灭菌的PCR管中向1μL稀释的Cas9中加入2.5μL sgRNA。混合后缓慢分配 sgRNA 超过 15 秒以防止沉淀。</li>
	<li>在室温下孵育 5 分钟以形成 RNP 复合物。</li>
</ol>5. 通过电穿孔递送 RNP
<ol><li>准备电穿孔尖端（比色皿）。按下柱塞以延长阀杆。将阀杆插入尖端。 注意： 确保尖端牢固，柱塞滑动平稳并超出尖端护套。如果吸头位置不正确，气泡可能会进入吸头，导致吸头故障。</li>
	<li>通过上下移液重悬细胞，然后用细胞加载电穿孔尖端。取出吸头的全部 10 μL 体积容量，避免起泡。</li>
	<li>从阴性对照 sgRNA 开始，从步骤 4 开始，将电穿孔尖端中的 10 μL 细胞转移到含有 3.5 μL RNP 的样品管中。上下移液三次，使混合均匀。将 10 μL 混合物吸回吸头，确保不存在气泡。</li>
	<li>将尖端放入电穿孔站，将其放入缓冲液中。避免用尖端接触塑料表面以保持无菌。在触摸屏显示器上按开始。 </li>
	<li>完成后，显示屏将指示脉冲成功。从设备中取出移液器，并将细胞置于标记的未包被的12孔板的干孔中。</li>
	<li>在 15 mL PBS + Ca/Mg 中上下移液两次冲洗吸头。确保尖端不接触任何东西并保持无菌。 注意：在许多实验中，可以将尖端重复用于多个样品，例如在无活性阴性对照sgRNA样品之后，或者在指导活性的初始评估期间，当每个基因测试四到五个sgRNA时，唯一的读数是编辑效率。然而，在对编辑过的细胞进行功能分析时，建议为每个基因使用新的提示，因为少量的sgRNA或细胞残留可能会影响实验结果。</li>
	<li>立即向细胞中加入 1 mL 无抗生素培养基（在步骤 3.6 中等分），轻轻摇动板混合。</li>
	<li>对其余反应重复步骤5.2-5.6。</li>
	<li>将细胞放回培养箱。</li>
	<li>电穿孔后1-2小时检查细胞的活力。细胞应>90%贴壁。可选：在电穿孔后 1-2 小时向细胞中加入庆大霉素 （5 μg/mL），以帮助防止污染，如果这不会干扰下游实验。</li>
</ol>6. 评估编辑效率
注意：大多数编辑在 48 小时后完成。
<ol><li>电穿孔后48小时检查细胞单层。如果细胞汇合度超过 50%，则继续。如果细胞汇合度<50%，请再等待1-2天。确定编辑效率的基因组 DNA 分析除了下游实验所需的细胞外，还需要 1 x 105 个细胞。</li>
	<li>制备基因组DNA（gDNA）。<ol><li>用PBS（-Ca/Mg）洗涤细胞。加入 1 mL PBS + 1 mM EDTA。在室温下孵育约5分钟，直到细胞开始从板上分离。</li>
		<li>通过移液分离细胞。转移到低蛋白结合微量离心管中。</li>
		<li>计数细胞并去除 1 x 105 个细胞的等分试样。剩余的细胞可以重新铺板用于进一步的实验。通常，实验在电穿孔后 5 天进行，以允许蛋白质衰变。</li>
		<li>在微量离心管中以最大速度沉淀细胞进行gDNA分析15秒。</li>
		<li>将沉淀重悬于50μL裂解缓冲液中。涡旋以分离粘附在管壁上的任何细胞，并在98°C下加热10分钟以裂解细胞并灭活内源性DNase。</li>
		<li>将试管放在冰上。</li>
		<li>加入 1 μL 蛋白酶 K （20 mg/mL）。在37°C孵育至少1小时;为了方便起见，它可以过夜。</li>
		<li>加热至98°C10分钟以灭活蛋白酶K。</li>
		<li>在室温下以最大速度离心5分钟。去除含有DNA的上清液。这种无需进一步纯化的粗制剂适用于大多数PCR反应。</li>
	</ol></li>
	<li>使用 Sanger 测序评估编辑效率。<ol><li>设计PCR引物，在最多5'的向导位点上游200-300 bp，在大多数3'的向导位点下游200-300 bp。典型的扩增子大小为 ~500 bp。设计一个巢式测序引物，距离最近的向导位点至少125 bp（图2A）。</li>
		<li>使用 2 μL gDNA 进行 PCR。</li>
	</ol></li>
	<li>准备用于测序的PCR产物。该协议使用核酸外切酶 I/虾碱性磷酸酶 （ExoI/SAP） 处理。商业 DNA 纯化试剂盒也可以使用，但需要更多的手动操作时间。<ol><li>制备 15 μL PCR 产物。加入 7.5 μL 水、1 μL 10x ExoI 缓冲液、10 U ExoI 和 1 U SAP 以降解干扰 Sanger 测序的 dNTP 和引物。</li>
		<li>在37°C孵育30分钟，然后在85°C孵育20分钟以使酶失活。</li>
	</ol></li>
	<li>对Exo/SAP处理的PCR产物进行Sanger测序;使用已知量的DNA大小标记来估计样品中存在的PCR产物的大小。测序反应可能需要优化，因为TIDE软件需要高质量的序列色谱图来准确估计编辑效率。未编辑位点的序列色谱图应提供具有最小背景的清晰峰（图2B，C）。</li>
	<li>为了估计编辑效率，使用TIDE使用编辑的gDNA和未编辑的对照gDNA分析Sanger测序色谱图文件。（表 1、 图 2）。上传 Sanger 测序文件并使用默认设置运行软件。</li>
</ol>

原代骨髓来源的巨噬细胞中精确基因破坏的指南

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W., Kim, S., Kim, J. M., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+engineering+in+human+cells+with+the+Cas9+RNA-guided+endonuclease.">Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.</a> Nature Biotechnology. 31 (3), 230-232 (2013).</li><li>Jinek, 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=RNA-programmed+genome+editing+in+human+cells.">RNA-programmed genome editing in human cells.</a> eLife. 2, e00471 (2013).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> eLife. 3, 04766 (2014).</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Research. 42 (22), e168 (2014).</li><li>Craft, J. <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-mediated+genome+editing+in+primary+murine+bone+marrow-derived+macrophages.">CRISPR-Cas9-mediated genome editing in primary murine bone marrow-derived macrophages.</a> University of California, Davis. , (2022).</li><li>Herb, M., Farid, A., Gluschko, A., Kr&#246;nke, M., Schramm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+primary+macrophages+with+in+vitro+transcribed+mRNA.">Highly efficient transfection of primary macrophages with in vitro transcribed mRNA.</a> Journal of Visualized Experiments. (153), e60143 (2019).</li><li>Yu, X., 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=Improved+delivery+of+Cas9+protein/gRNA+complexes+using+lipofectamine+CRISPRMAX.">Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.</a> Biotechnology Letters. 38 (6), 919-929 (2016).</li></ol>

作者没有什么可透露的。

使用电穿孔Cas9-sgRNA复合物进行基因组编辑可以有效破坏BMDM中的基因功能。编辑效率因靶序列和基因而异。通常，通常会筛选四到五个 sgRNA，以鉴定出一种高活性的 sgRNA。一些基因座的编辑效率较低，很可能是由于染色质结构。在这些情况下，可以进行一些修改以提高编辑效率。将两个活性 sgRNA 共同递送至同一外显子可改善某些基因的编辑。然而，当两个向导共转染时，我们观察到 TIDE 可能会失?...

巨噬细胞是先天免疫细胞，在组织修复和免疫中起关键作用 1,2。永生化的巨噬细胞系，如小鼠 RAW 264.7 细胞或人 THP-1 细胞，具有多种有益特性，包括通过递送 RNA 干扰或 CRISPR/Cas9 载体来促进生长和易于基因破坏 3,4。然而，致癌转化会显着改变它们的生理机能，这导致某些通路的异常激活和其他通路的沉默反应 5,6。原代骨髓来源的巨噬细胞 （BMDM） 更接近地概括了体内巨噬细胞的生理学，但由于这些原代免疫细胞中的质粒转染和病毒转导效率低，因此在遗传操作方面仍然具有挑战性 7,8。因此，需要更有效的方法来破坏基因功能。
CRISPR/Cas9 基因组编辑是跨一系列生物系统（包括哺乳动物细胞9、10、11、12）进行遗传操作的强大工具。化脓性链球菌 Cas9 蛋白与序列特异性向导 RNA 复合时可有效且特异性地切割双链 DNA。通过裂解 DNA 的非同源末端连接 （NHEJ） 进行 DNA 修复会导致小的插入或缺失（插入缺失），从而产生移码突变。在早期研究中，Cas9 和 sgRNA 通过质粒或慢病毒载体递送，这是许多细胞系的有效递送方法 9,10。然而，由于通过转染或转导递送载体的效率低，原代细胞，特别是原代免疫细胞通常对这些方法无效。随后，已经开发了在体外生成 sgRNA-Cas9 复合物并通过电穿孔递送它们的方法，这些方法在多种细胞类型中实现了高效率13,14。结果表明，使用这种方法在原代巨噬细胞中进行基因组编辑的可能性。
在这里，我们提供了一种使用 sgRNA-Cas9 核糖核蛋白复合物 （RNP） 在初级 BMDM 中进行基因组编辑的方案。它包含减轻原代免疫细胞中存在的免疫传感器激活的步骤，并以最小的毒性在靶向位点进行高达 95% 的编辑。该协议还包括使用常规聚合酶链反应 （PCR） 和 Sanger 测序评估编辑效率的工作流程，然后通过分解跟踪插入缺失 （TIDE）15（一种经过充分验证的在线软件工具）进行计算机分析。

<table><tbody><tr><td>3T3-MCSF Cell Line</td><td>Gift from Russell Vance</td><td>not applicable</td><td></td></tr><tr><td>Alt-R Cas9 Electroporation Enhancer</td><td>IDT</td><td>1075915</td><td></td></tr><tr><td>Ampure XP Reagent Beads</td><td>Beckman Coulter</td><td>A63880</td><td></td></tr><tr><td>Calf intestinal alkaline phosphatase</td><td>NEB</td><td>M0525S</td><td></td></tr><tr><td>DNase</td><td>NEB</td><td>M0303S</td><td></td></tr><tr><td>DPBS +Ca/Mg (0.9mM CaCl2 and 0.5mM MgCl2)</td><td>Thermo Fisher</td><td>14040-133</td><td></td></tr><tr><td>DPBS -Ca/Mg</td><td>Thermo Fisher</td><td>14190-144</td><td></td></tr><tr><td>ExoI</td><td>NEB</td><td>M0293S</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Corning</td><td>35-015-CV</td><td></td></tr><tr><td>Herculase DNA polymerase &amp;amp; buffer</td><td>Agilent</td><td>600677</td><td></td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit</td><td>NEB</td><td>E2040S</td><td></td></tr><tr><td>LoBind conical tubes 15 mL</td><td>Eppendorf</td><td>30122216</td><td></td></tr><tr><td>LoBind Eppendorf tubes 2 mL</td><td>Eppendorf</td><td>22431102</td><td></td></tr><tr><td>NEBuffer r2.1</td><td>NEB</td><td>B6002S</td><td></td></tr><tr><td>Neon Transfection System</td><td>Thermo Fisher</td><td>MPK5000, MPP100, MPS100</td><td></td></tr><tr><td>Neon Transfection System 10 uL Tips</td><td>Thermo Fisher</td><td>MPK1025 or MPK1096</td><td></td></tr><tr><td>PBS + 1mM EDTA</td><td>Lonza</td><td>BE02017F</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Fisher</td><td>EO0491</td><td></td></tr><tr><td>rCutSmart Buffer for ExoI</td><td>NEB</td><td>B6004S</td><td></td></tr><tr><td>Ribolock</td><td>Thermo Fisher</td><td>EO0384</td><td></td></tr><tr><td>RNA loading dye</td><td>NEB</td><td>B0363S</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>S. pyogenes Cas9-NLS</td><td>University of California Macro Lab</td><td>not applicable</td><td>Available to non-UC investigators through&amp;nbsp; https://qb3.berkeley.edu</td></tr><tr><td>S. pyogenes Cas9-NLS, modified 3rd Generation</td><td>IDT</td><td>1081059</td><td></td></tr><tr><td>SAP</td><td>NEB</td><td>M0371S</td><td></td></tr></tbody></table>

high-efficiency gene disruption in primary bone marrow-derived macrophages using electroporated cas9-sgrna complexes

来自小鼠的骨髓来源的巨噬细胞（BMDM）是研究组织巨噬细胞复杂生物学的关键工具。作为原代细胞，它们比永生化的巨噬细胞系更接近 于体内 巨噬细胞的生理学模型，并且可以从已经携带特定遗传变化的小鼠中提取。然而，破坏BMDM的基因功能在技术上仍然具有挑战性。在这里，我们提供了一种在BMDM中进行高效CRISPR / Cas9基因组编辑的协议，该协议允许引入小的插入和缺失（插入缺失），从而导致破坏基因功能的移码突变。该方案描述了如何合成单向导RNA（sgRNA-Cas9）并形成纯化的sgRNA-Cas9核糖核蛋白复合物（RNP），这些复合物可以通过电穿孔递送。它还提供了一种使用常规 Sanger 测序和免费提供的在线分析程序来监控编辑效率的有效方法。该方案可在1周内进行，不需要质粒构建;它通常会导致 85% 到 95% 的编辑效率。

IVT模板是127 bp PCR产物（图1B）。全长 IVT 产物是 98 nt RNA，其迁移类似于 70 bp 双链 DNA 片段（图 1C）。
电穿孔后，细胞的存活率应为 >90%，总细胞计数为起始细胞数的 >70%。由此产生的突变细胞池应该具有一组不同的插入缺失，从Cas9切割位点附近开始。通过PCR和Sanger测序对靶基因的分析应显示Cas9切割位点下游每个位置的多个核苷酸?...

Watch this Scientific Journal Video about Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption at JoVE.com

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

该协议描述了使用 体外 组装并通过电穿孔递送的Cas9-sgRNA核糖核蛋白复合物在小鼠骨髓来源的巨噬细胞中进行基因组编辑的过程。

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 ...

我们的实验室研究分枝杆菌、结核病和宿主免疫系统之间的相互作用，重点是细菌和巨噬细胞之间的初始相互作用。巨噬细胞中使用了多种基因破坏技术，包括RNAi技术和CRISPR技术。主要挑战之一是巨噬细胞中基因破坏的低效率。该方案能够快速有效地对原代巨噬细胞进行遗传操作，而无需克隆和质粒构建。这些方法应该广泛适用于免疫学，使研究人员能够破坏原代巨噬细胞中的基因，以进行广泛的研究。

high-efficiency-gene-disruption-primary-bone-marrow-derived

Universidad San Sebastian

Research

JoVE Journal

Genetics

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

1.4K 次观看.  University of California, Davis. 我们的实验室研究分枝杆菌、结核病和宿主免疫系统之间的相互作用，重点是细菌和巨噬细胞之间的初始相互作用。巨噬细胞中使用了多种基因破坏技术，包括RNAi技术和CRISPR技术。主要挑战之一是巨噬细胞中基因破坏的低效率。该方案能够快速有效地对原代巨噬细胞进行遗传操作，而无需克隆和质粒构建。这些方法应该广泛适用于免疫学，使研究人员能够破坏原代巨噬...

视频: 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

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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 ...

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. ...

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

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

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 ...

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 ...

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

<meta charset="utf8"></head><body>CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical ...