Name: development of knock-out muscle cell lines using lentivirus-mediated crispr/cas9 gene editing
Uploaded: 2022-06-16T14:30:00
Description: Watch this Scientific Journal Video about Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing at JoVE.com

这项工作由法国肌病协会（AFM-Téléthon）和奥弗涅-罗纳阿尔卑斯大区（AURA）的赠款资助。

肌肉活检是根据欧洲建议和法国立法从研究组织银行（Myobank，欧盟网络EuroBioBank的合作伙伴，法国巴黎）获得的。已获得所有人的书面知情同意。永生化成肌母细胞由V. Mouly博士（法国巴黎Myology Institute）友好地制作，并且协议由Myology Institute伦理委员会（MESRI，n AC-2019-3502）批准。
1. 防鲜膜导轨设计
<ol><li>确定要删除的基因区域。使用基因组浏览器工具（如 ensem...

<ol>
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在表征涉及病理学的未知功能基因的道路上，一个重要的步骤是开发相关的细胞模型来研究这些基因的功能。使用CRISPR / Cas9进行基因编辑是一个呈指数级增长的研究领域，这里介绍的敲除模型的开发是其使用最广泛的应用之一。在这种情况下，我们在这里提出了一种多功能方案，可以在任何感兴趣的基因中开发一种人类细胞系敲除，从而允许在相关的人类细胞模型中表征该基因。这里介绍的方案...

随着基因测序的进展和特定组织中未知功能基因突变的鉴定，开发相关细胞模型以了解新靶基因的功能并确认其参与相关病理生理学机制构成了必不可少的工具。此外，这些模型对未来的治疗发展1，2具有重要意义，并且构成了与国际上关于减少在实验中使用动物的建议直线开发敲除动物模型的有趣替代方案。使用CRISPR / Cas9进行基因编辑是目前最强大的工具之一，它允许开发许多敲除/敲入模型，并且使用CRISPR / Cas9进行靶向基因验证是CRISPR / Cas93使用最广泛的应用之一。基因编辑的成功依赖于在靶细胞模型中引入CRISPR工具（引导RNA和核酸酶Cas9）的能力，这在许多难以转染的细胞（如肌肉细胞4）中可能是一个挑战。这种挑战可以通过使用病毒（通常是慢病毒）来克服，其具有有效转译许多细胞类型并递送其转基因的巨大优势。但它的主要缺点是转基因在宿主细胞基因组中的整合，导致定位在整合位点的基因的潜在改变和转基因的永久表达，这在核酸酶Cas9的情况下会导致破坏性后果5。Merienne及其同事6提出了一种智能解决方案，其中包括将靶向Cas9基因本身的引导RNA引入细胞，导致Cas9失活。本文将这种策略的适应性作为用户友好和多功能的方案提出，允许敲除难以转染的细胞中的几乎任何基因。
这里提出的方案的目标是诱导永生化肌肉细胞中目的基因的失活。它可用于敲除不同类型的永生化细胞中的任何目的基因。这里描述的方案包含设计指导RNA并将其克隆成慢病毒质粒的步骤，在慢病毒载体中生产CRISPR工具，用不同的慢病毒转导细胞，以及克隆细胞以产生均匀的编辑细胞系的步骤。
使用该协议，已经开发了永生化的人骨骼肌细胞，并删除了1型ryanodine受体（RyR1），这是一种参与细胞内钙释放和肌肉收缩的必需钙通道7。该基因的敲除（KO）已使用蛋白质印迹在蛋白质水平上得到证实，并且在功能水平上使用钙成像得到证实。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-CACNA1S antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA048892</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Blp I</td>
			<td>NE BioLabs</td>
			<td>R0585S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>CalPhos Mammalian Transfection Kit</td>
			<td>Takara</td>
			<td>631312&#160;</td>
			<td>Transfection kit</td>
		</tr>
		<tr>
			<td>Easy blot anti Mouse IgG</td>
			<td>GeneTex</td>
			<td>GTX221667-01</td>
			<td>HRP secondary antibody</td>
		</tr>
		<tr>
			<td>Easy blot anti Rabbit IgG</td>
			<td>GeneTex</td>
			<td>GTX221666</td>
			<td>HRP secondary antibody</td>
		</tr>
		<tr>
			<td>Fluo-4 direct</td>
			<td>Molecular Probes</td>
			<td>F10472</td>
			<td>Calcium imaging</td>
		</tr>
		<tr>
			<td>GAPDH(14C10) Rabbit mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>#2118</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>Fermentas</td>
			<td>ER0501</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>InFusion HD Precision Plus</td>
			<td>Takara</td>
			<td>638920</td>
			<td>Ligation kit</td>
		</tr>
		<tr>
			<td>MasterMix Phusion High Fidelity with GC</td>
			<td>ThermoFisher Scientific</td>
			<td>F532L</td>
			<td>Mix for PCR reaction with High fidelity Taq polymerase and dNTPs</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain antibody</td>
			<td>DHSB</td>
			<td>MF20</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi EF</td>
			<td>Macherey-Nagel</td>
			<td>REF&#160;740424</td>
			<td>Maxipreparation kit for purification of plasmids</td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue</td>
			<td>Macherey-Nagel</td>
			<td>740952</td>
			<td>Kit for DNA extraction from cell</td>
		</tr>
		<tr>
			<td>One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td>ThermoFisher Scientific</td>
			<td>C737303</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>Plasmid #87904</td>
			<td>Addgene</td>
			<td>87904</td>
			<td>Lentiviral plasmid encoding the SpCas9 (for LV-Cas9)</td>
		</tr>
		<tr>
			<td>Plasmid #87919</td>
			<td>Addgene</td>
			<td>87919</td>
			<td>Lentiviral backbone for insertion of cassette with guides (for LV-guide-target)</td>
		</tr>
		<tr>
			<td>Plasmid #12260</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Lentiviral plasmid encoding lentiviral packaging GAG POL</td>
		</tr>
		<tr>
			<td>Plasmid #8454</td>
			<td>Addgene</td>
			<td>8454</td>
			<td>Lentiviral plasmid encoding envelope protein for producing lentiviral and MuLV retroviral particles</td>
		</tr>
		<tr>
			<td>V5 Tag Monoclonal Antibody</td>
			<td>Invitrogene</td>
			<td>R96025&#160;</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>XL10-Gold Ultracompetent Cells</td>
			<td>Agilent</td>
			<td>200317</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>Xma I</td>
			<td>NE BioLabs</td>
			<td>R0180S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

development of knock-out muscle cell lines using lentivirus-mediated crispr/cas9 gene editing

簇状调节间隔短回文重复序列（CRISPR）/Cas 9的一个重要应用是敲除细胞系的开发，专门用于研究在基因诊断期间鉴定的与疾病相关的新基因/蛋白质的功能。对于这种细胞系的开发，必须解开两个主要问题：将CRISPR工具（Cas9和引导RNA）高效插入所选细胞中，以及将Cas9活性限制在所选基因的特定缺失上。这里描述的方案专门用于将CRISPR工具插入难以转染的细胞中，例如肌肉细胞。该协议基于使用慢病毒，用公开可用的质粒生产，其所有克隆步骤都被描述为靶向目的基因。Cas9活性的控制是使用先前描述的称为KamiCas9的系统进行的，其中用编码靶向Cas9的引导RNA的慢病毒转导细胞允许逐步消除Cas9表达。该方案已应用于 RYR1-敲除人肌肉细胞系的开发，该细胞系已在蛋白质和功能水平上进一步表征，以确认参与肌肉细胞内钙释放和激发 - 收缩耦合的这种重要钙通道的敲除。这里描述的过程可以很容易地应用于肌肉细胞或其他难以转染的细胞中的其他基因，并产生有价值的工具来研究人类细胞中的这些基因。

该协议应用于来自健康受试者15 的永生化成肌母细胞（所谓的HM细胞，用于人类成肌细胞），其中RyR1先前已被表征为16，以敲除编码 RyR1 蛋白的RYR1基因。指南RNA的设计是为了删除包含基因外显子101和内含子101部分的序列。预计删除部分外显子101将导致阅读框的中断。此外，外显子101编码蛋白质的孔，因此需要产生功能性钙通道。许多患有严重RyR1相关肌病...

Watch this Scientific Journal Video about Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing at JoVE.com

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

该协议描述了如何使用CRISPR / Cas9生成敲除肌母细胞，从引导RNA的设计到细胞克隆和敲除克隆的表征。

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, ...

该协议允许在任何基因中开发肌肉细胞敲除，以进一步研究该基因在肌肉中的功能。优点是它比研究任何基因的敲除动物的发育便宜，更快。例如，这种技术可用于研究新发现的基因在肌病中的参与。如果将这里描述的技术应用于iPS细胞，它可能导致任何类型的细胞中任何基因的敲除细胞的发展。要开始成簇的有规律间隔的短回文重复序列或CRISPR介导的基因缺失，首先使用基因组浏览器工具，如ensembl。org来鉴定靶基因及其两侧的DNA序列。要获得基因外显子的列表，首先单击蛋白质编码序列的转录本，然后单击外显子。接下来，点击下载序列，仅选择基因组序列，下载整个基因的完整共识序列。滚动基因的外显子和内含子列表以选择靶向的外显子和内含子。为了设计引导RNA，首先选择靶序列上游和下游内含子的核苷酸序列。然后去一个网站，如crispr.tefor。网并使用选定的序列来设计两个引导RNA，每个RNA长20个核苷酸，没有原始间隔相邻基序，由几百个碱基对隔开。接下来确定每个引导RNA的反向补体序列并对引物进行排序。为了构建用于质粒克隆的指导RNA盒，首先使用文本中描述的配方和程序使用一组引物运行PCR，然后使用Tris-borate-EDTA缓冲液在1%琼脂糖凝胶中分离PCR产物。接下来切除300碱基对碎片并使用试剂盒纯化。同样，在用另一组引物运行另一个PCR后，将400碱基对长的DNA片段纯化成20微升的洗脱缓冲液。要将引导RNA盒插入慢病毒质粒主链中，首先按照文中所述设置质粒与限制性内切酶Xma 1和Blp 1的双重消化反应。将反应管在37摄氏度孵育一小时后，将其在65摄氏度孵育20分钟，然后在1%琼脂糖凝胶中运行消化产物，并使用适当的试剂盒纯化约10千碱基对的质粒。还可以通过测量光密度来量化纯化产物。将引导RNA盒与质粒结合，与纯化的引导RNA，线性化的质粒DNA，酶和水制备反应混合物至最终体积为10微升，然后将反应管在50摄氏度下孵育15分钟以产生最终质粒，称为pGuide。对于慢病毒生产，首先在DMEM中用100万HEK 293细胞的145厘米板接种，其中含有高葡萄糖和丙酮酸，并补充10%FBS和1%青霉素或链霉素。然后将细胞在37摄氏度下在5%二氧化碳培养箱中生长三天。第四天检查细胞以确保60%至65%的汇合度。接下来制备由目标质粒、编码病毒包膜的质粒、慢病毒包装质粒psPAX2和磷酸钙组成的转染溶液。用水将最终体积调节至1000微升。然后在搅拌下将转染溶液滴加到一毫升2X HBS中，并在室温下孵育至少10分钟。之后，将溶液滴加到细胞中。对于转染溶液的均匀分布，轻轻地向所有方向倾斜板，然后将细胞在37摄氏度下在5%二氧化碳培养箱中孵育至少五个小时。接下来，从平板中取出培养基并用PBS洗涤细胞以除去转染试剂，然后加入12毫升新鲜培养基。在37摄氏度下孵育48小时后，将所有板中的培养基池化到管中。将管放入密封桶中，在4摄氏度下以800倍G离心5分钟，然后使用0.45微米过滤器过滤上清液，并使用摆动桶转子将滤液以68，300倍G离心两小时，在4摄氏度下。除去上清液后，将管子倒置在安全柜中的纸巾上5至10分钟，以最大程度地从颗粒中除去液体。加入100微升HEC增殖培养基，并将管保持在四摄氏度至少两小时。然后通过移液重悬沉淀。收集所有重悬的颗粒后，制作10或25微升的等分试样，然后在液态氢中速冻后将等分试样储存在零下80摄氏度。对于成肌细胞转导，首先用100微升含有10， 000个成肌细胞的增殖培养基接种96孔板的每个孔。第二天，将预先计算的左心室导线和左心室Cas9体积添加到安全柜中的电池中。孵育五天后，通过添加胰蛋白酶从孔中释放细胞。计数细胞数量后，将细胞从具有40%至50%汇合度的孔转移到新板上。孵育 5 小时后，在感染的多重程度 20 时加入计算体积的左心室杀手，并孵育 5 至 10 天或介于两者之间至少两次传代。为了通过钙成像对基因编辑克隆进行功能表征，在层粘连蛋白包被的35毫米培养皿的中心板上放置50， 000个细胞。要诱导分化，请添加文中描述的分化培养基。孵育六天后，取下培养基并用疏水笔描绘细胞。然后向分化培养基中加入50微升的Fluo-4 Direct，一对一稀释到分化的肌管中。将培养皿在37摄氏度下孵育30分钟后，用Krebs缓冲液洗涤细胞两次，并补充每毫升葡萄糖一毫克。接下来使用倒置荧光显微镜或具有10X物镜的共聚焦显微镜以每秒一帧的速率测量荧光变化90秒，并选择至少具有10个肌管的场。除去额外的克雷布斯缓冲液后，用两毫升氯化钾刺激细胞进行膜去极化或两毫升氟氯间甲酚或4CmC或RyR1直接刺激。可视化刺激后的荧光变化，然后量化每个肌管中的荧光变化。该协议可以成功地用于从人类成肌细胞中敲除RyR1基因，这导致细胞中的基因组DNA更短。RyR1敲除也在蛋白质水平上得到证实，因为RyR1蛋白无法通过蛋白质印迹在靶细胞中检测到。通过4CmC直接RyR1刺激或氯化钾诱导膜去极化间接刺激后，通过Fluo-4钙成像进一步证实了与RyR1敲除细胞分化的肌管中RyR1活性的缺乏。将要删除的序列应该是功能蛋白预测所必需的。该技术非常适合开发基因组后工具，以研究新基因在肌肉疾病中的参与。

Research

JoVE Journal

Genetics

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

科研

遗传学

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

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 ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

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. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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 ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

通过CAS9蛋白-gRNA复合物电穿孔直接基因敲除Axolotl脊髓神经干细胞

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...