Name: construction of crispr plasmids and detection of knockout efficiency in mammalian cells through a dual luciferase reporter system
Uploaded: 2020-12-05T12:30:00
Description: Watch this Scientific Journal Video about Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System at JoVE.com

该项目由山东省一级草原科学学科计划、国家自然科学基金（31301936）资助。 31572383、公益农业科学研究专项基金（201403071）、国家奶制品质量安全风险评估重大专项（GJFP201800804）和青岛市民生科技项目（19-6-1-68-nsh， 14-2-3-45-nsh，13-1-3-88-nsh）。

1. sgRNA寡核苷酸设计
<ol><li>使用在线工具（如 Cas-Designer 在线工具（http://www.rgenome.net/cas-designer/） 设计 sgRNA。基于使用的 Cas9，PAM 序列非常重要。对于 xCas9，相关的 PAM 序列是 NG，前引用的 Cas-Designer 在线工具可以生成 xCas9 相关的 sgRNA。<ol>
<li>使用 sgRNA 设计工具，这些工具包含目标上和目标外预测 （http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design）11的算法。首选分数为 0....

<ol>
	<li>Zhang, H., 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=A+surrogate+reporter+system+for+multiplexable+evaluation+of+CRISPR/Cas9+in+targeted+mutagenesis.">A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis.</a> Scientific Reports. 8 (1), 1042 (2018).</li><li>Nageshwaran, S., 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=CRISPR+Guide+RNA+Cloning+for+Mammalian+Systems.">CRISPR Guide RNA Cloning for Mammalian Systems.</a> Journal of Visualized Experiments. (140), (2018).</li><li>Dang, Y., 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=Optimizing+sgRNA+structure+to+improve+CRISPR-Cas9+knockout+efficiency.">Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.</a> Genome Biology. 16, 280 (2015).</li><li>Doench, J. G., 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=Optimized+sgRNA+design+to+maximize+activity+and+minimize+off-target+effects+of+CRISPR-Cas9.">Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.</a> Nature Biotechnology. 34 (2), 184-191 (2016).</li><li>Moreno-Mateos, M. A., 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=CRISPRscan:+designing+highly+efficient+sgRNAs+for+CRISPR-Cas9+targeting+in+vivo.">CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.</a> Nature Methods. 12 (10), 982-988 (2015).</li><li>Joung, J., 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=Genome-scale+CRISPR-Cas9+knockout+and+transcriptional+activation+screening.">Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.</a> Nature Protocols. 12 (4), 828-863 (2017).</li><li>Yang, L., Yang, J. L., Byrne, S., Pan, J., Church, G. M. <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-Directed+Genome+Editing+of+Cultured+Cells.">CRISPR/Cas9-Directed Genome Editing of Cultured Cells.</a> Current Protocols in Molecular Biology. 107, 1-17 (2014).</li><li>Yang, L., Mali, P., Kim-Kiselak, C., Church, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas-mediated+targeted+genome+editing+in+human+cells.">CRISPR-Cas-mediated targeted genome editing in human cells.</a> Methods in Molecular Biology. 1114, 245-267 (2014).</li><li>Vidigal, J. A., Ventura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+one-step+generation+of+paired+gRNA+CRISPR-Cas9+libraries.">Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries.</a> Nature Communications. 6, 8083 (2015).</li><li>Mazon, G., Mimitou, E. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Homologous+recombination+in+DNA+double-strand+break+repair.">SnapShot: Homologous recombination in DNA double-strand break repair.</a> Cell. 142 (4), 646 (2010).</li><li>Doench, J. G., 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=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nature Biotechnology. 32 (12), 1262-1267 (2014).</li><li>Lee, J. K., 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=Directed+evolution+of+CRISPR-Cas9+to+increase+its+specificity.">Directed evolution of CRISPR-Cas9 to increase its specificity.</a> Nature Communications. 9 (1), 3048 (2018).</li><li>Sung, Y. H., 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=Highly+efficient+gene+knockout+in+mice+and+zebrafish+with+RNA-guided+endonucleases.">Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.</a> Genome Research. 24 (1), 125-131 (2014).</li><li>Ran, F. A., 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=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154 (6), 1380-1389 (2013).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (12), 5463-5467 (1977).</li><li>Ruan, J., 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=Highly+efficient+CRISPR/Cas9-mediated+transgene+knockin+at+the+H11+locus+in+pigs.">Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs.</a> Scientific Reports. 5, 14253 (2015).</li><li>Li, K., 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=An+plasmid+with+double+fluorescent+groups+and+its+application+as+standard+substance.">An plasmid with double fluorescent groups and its application as standard substance.</a> China patent. , (2012).</li><li>Li, H., 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=Characterization+of+the+porcine+p65+subunit+of+NF-kappaB+and+its+association+with+virus+antibody+levels.">Characterization of the porcine p65 subunit of NF-kappaB and its association with virus antibody levels.</a> Molecular Immunology. 48 (6-7), 914-923 (2011).</li><li>Li, H., 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=A+pair+of+sgRNAs+targeting+porcine+RELA+gene.">A pair of sgRNAs targeting porcine RELA gene.</a> China patent. , (2015).</li></ol>

我们在此描述的 sgRNA 载体克隆程序有助于高效生产 sgRNA，大部分成本来自寡核苷酸排序和载体测序。虽然概述的方法旨在允许用户生成用于CRISPR/Cas9的sgRNA，但该协议可以很容易地适用于Cas9正交器或其他RNA引导的内核，如Cpf1，对载体骨干和寡核苷酸悬垂序列进行细微的修改。
上述协议将在10天内提供首选的sgRNA靶点，当从适当设计的寡核苷酸开始。这包括 sgRNA 设计 （1 小时?...

CRISPR sgRNA由20核苷酸序列（原空间）组成，是对基因组靶序1，2的补充。虽然效率很高，但CRISPR/Cas系统修改给定基因组位点的能力需要生成携带目标位点独一无二的高效sgRNA的载体。本文介绍了生成该 sgRNA 载体的关键步骤。
对于使用CRISPR/Cas系统成功编辑基因组，使用高效的sgRNA是一个关键的先决条件3，4，5。由于用于基因组编辑的工程核酸酶在不同靶点1上表现出不同的效率，因此为了节省时间和精力，有必要预先选择候选sgRNA靶点。已开发出双荧光素酶报告系统，通过检查双链断裂修复通过单股退火3，10来评估淘汰效率。在这里，我们使用这个记者系统从不同的候选sgRNA载体中选择一个首选的CRISPR sgRNA靶点，这些载体设计用于特定的基因编辑。此处所述的协议已在我们的小组和协作实验室中实施，以生成和评估 CRISPR sgRNA。
以下协议总结了如何通过网络软件设计出合适的sgRNA。选择合适的 sgRNA 后，我们描述了获取所需寡核苷酸的不同步骤，以及将配对寡核苷酸插入 pX330-xCas9 表达载体的方法。我们还提出了一种基于这些序列的连体到预消化的表达向量（步骤2-10，图1A）的基础上组装sgRNA表达和双荧光 素酶报告器向量的方法。最后，我们描述了如何分析每个sgRNA的DNA切割效率（步骤11-12）。

<table>
	<tbody>
		<tr>
			<td>A new generation of full touch screen gradient PCR instrument</td>
			<td>LongGene</td>
			<td>A200</td>
			<td>Target gene amplification</td>
		</tr>
		<tr>
			<td>AscI restriction enzymes</td>
			<td>New England Biolabs</td>
			<td>R0558V</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>BbsI restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>Clean workbench</td>
			<td>AIRTECH</td>
			<td>SW-CJ-2FD/VS-1300L-U</td>
			<td>A partial purification device in the form of a vertical laminar flow, which creates a local high clean air environment</td>
		</tr>
		<tr>
			<td>DH5&#945; Competent Cells</td>
			<td>TaKaRa</td>
			<td>K613</td>
			<td>Plasmid vector transformation</td>
		</tr>
		<tr>
			<td>Dual-Luciferas Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>Dual-luciferas reporter assay</td>
		</tr>
		<tr>
			<td>Electric thermostatic water bath</td>
			<td>Sanfa Scientific Instruments</td>
			<td>DK-S24</td>
			<td>Heating reagent by constant temperature in water bath</td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td>Beijing Liuyi Biotechnology Co., Ltd.</td>
			<td>DYY-6C</td>
			<td>Control voltage, current, etc.</td>
		</tr>
		<tr>
			<td>Eppendorf Reference 2</td>
			<td>Eppendorf China Ltd.</td>
			<td>Reference 2</td>
			<td>Accurately draw and transfer traces of liquid</td>
		</tr>
		<tr>
			<td>Gel imaging analyzer</td>
			<td>Beijing Liuyi Biotechnology Co., Ltd.</td>
			<td>WD-9413B</td>
			<td>For the analysis of electrophoresis gel images</td>
		</tr>
		<tr>
			<td>GloMax 20/20 Luminometer</td>
			<td>Promega</td>
			<td>E5311</td>
			<td>Detect dual luciferase activity</td>
		</tr>
		<tr>
			<td>High speed refrigerated centrifuge</td>
			<td>BMH</td>
			<td>sigma 3K15</td>
			<td>Nucleic acid extraction and purification</td>
		</tr>
		<tr>
			<td>Intelligent biochemical incubator</td>
			<td>Sanfa Scientific Instruments</td>
			<td>SHP-160</td>
			<td>Provide a suitable temperature environment for the enzyme digestion experiment</td>
		</tr>
		<tr>
			<td>LB Broth Agar</td>
			<td>Sangon Biotech</td>
			<td>A507003-0250</td>
			<td>For the cultivation of E.coli</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent Kit</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td>DNA Transfection</td>
		</tr>
		<tr>
			<td>SalI restriction enzymes</td>
			<td>New England Biolabs</td>
			<td>R3138V</td>
			<td>Cutting target vectors</td>
		</tr>
		<tr>
			<td>SanPrep Column DNA Gel Extraction Kit</td>
			<td>Sangon Biotech</td>
			<td>B518131-0050</td>
			<td>Recycling DNA fragments</td>
		</tr>
		<tr>
			<td>SanPrep Column Plasmid Mini-Preps Kit</td>
			<td>Sangon Biotech</td>
			<td>B518191-0100</td>
			<td>Extraction of plasmid DNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202V</td>
			<td>Link DNA fragment</td>
		</tr>
		<tr>
			<td>TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0</td>
			<td>TaKaRa</td>
			<td>9761</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>Vertical pressure steam sterilizer</td>
			<td>JIBIMED</td>
			<td>LS-50LD</td>
			<td>High temperature and autoclave to kill bacteria, fungi and other microorganisms in laboratory equipment</td>
		</tr>
		<tr>
			<td>Water bath thermostat</td>
			<td>Changzhou Guoyu Instrument Manufacturing Co., Ltd.</td>
			<td>SHZ-82</td>
			<td>Let the bacteria keep shaking, which is good for contact with air.</td>
		</tr>
	</tbody>
</table>

construction of crispr plasmids and detection of knockout efficiency in mammalian cells through a dual luciferase reporter system

虽然效率很高，但由CRISPR酶对基因组位点进行修饰需要事先生成目标位点特有的sgRNA。这项工作描述了导致构建高效 sgRNA 载体的关键步骤，该策略允许在 DNA 测序之前通过 PCR 有效检测正菌落。由于使用CRISPR系统进行有效的基因组编辑需要高效的sgRNA，因此必须预选候选sgRNA目标，以节省时间和精力。开发了双荧光素酶报告机系统，通过检查通过单股退火检查双链断裂修复来评估敲除效率。在这里，我们使用这个记者系统从候选sgRNA载体中选取首选的xCas9/sgRNA靶点进行特定基因编辑。概述的协议将在10天内提供首选的sgRNA/CRISPR酶载体（从适当设计的寡核苷酸开始）。

本协议中概述的方法用于构建sgRNA和xCas9表达载体，然后对具有相对较高的基因靶向效率的sgRNA寡基因进行优化筛选。在这里，我们展示了一个代表性的例子，3个sgRNA目标羊 DKK2 exon 1。SgRNA 和 xCas9 表达载体可以通过预消化向量骨干 （图2） 构建，然后通过退火寡头对将载体与一系列短的双链 DNA 片段连在一起。正菌落可以通过特定的底向对引导PCR检测（?...

Watch this Scientific Journal Video about Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System at JoVE.com

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

在这里，我们提出了一个协议，描述了一个简化的方法，用于有效生成质粒，同时表达CRISPR酶和相关的单导RNA（sgRNA）。哺乳动物细胞与这种sgRNA/CRISPR载体的共转染，以及检查双链断裂修复的双荧光素酶报告器载体，可以评估敲除效率。

通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

该协议描述了生成和优化高效 sgRNA 载体的关键步骤。我们对候选 sgRNA 的预加速针对相对时间和推论。该协议的主要优点是，它使得分析每个sgRNA的DNA编码印象，而无需编辑内源基因。这种预选择方法也可以通过双链断裂应用于其他基因编辑措施。首先将冻干寡核苷酸稀释到双蒸馏水中的最终浓度为10微摩尔。在薄壁PCR管中以一比一的比例混合前进和反向寡核苷酸，无需添加任何额外的缓冲液。在95摄氏度下孵育混合物5分钟，然后将温度降到72摄氏度10分钟。从 PCR 机器上去除寡核苷酸混合物，并允许它在室温下冷却。要消化pX330-xCas9载体，与Bbs1一起，将载体、酶和消化缓冲液在50微升体积中结合，并在37摄氏度下孵育反应两小时。进行细胞转化后，从 LB 板中选择 5 到 10 个细菌菌落，并在 1.5 毫升管中使用 1 毫升 LB 培养基接种 1 毫升 LB 介质。然后在旋转摇床上孵育管子两到三个小时。执行 PCR 以筛选文本手稿中描述的重组质粒，然后在每厘米 10 伏以下的 2% Agarose 凝胶上运行产品。识别阳性质粒后，使用它们以及记者向量共同感染适当的细胞系。要对细胞进行精使，请从培养的细胞中去除生长介质。用 PBS 轻轻清洗培养容器的表面，将 100 微升 PLB 放入每个培养井中，以完全覆盖细胞单层。让板在室温下孵育20分钟，然后将落酸转移到管或小瓶中，以进行进一步处理或储存。要执行双荧光素酶测定，对亮度计进行编程，以提供两秒的预读延迟，然后进行 10 秒的测量周期。然后，将100微升荧光素测定试剂分配到适当数量的发光计管中。小心地将20微升的细胞利沙酸盐加入发光计管中，通过移液混合两到三次。将管子放在发光计中并启动读数。从发光仪上拆下样品管。加入100微升停止试剂和涡流短暂混合。将样品返回亮度计并开始读数。记录蕾妮拉路西法酶活动，与萤火虫路西法酶活动正常化，特别是屏幕上显示的比例的倒数。完成后丢弃反应管。该方法用于为绵羊DKK2 Exon 1生成三个sgRNA载体。sgRNA 和 xCas9 表达载体是通过预先消化向量骨干，然后通过退火寡对将载体骨干连接成一系列短的双链 DNA 片段而构建的。用特定的底基因对引导PCR对筛选了7个细菌菌落，并检测出阳性菌落。通过双荧光素酶测定同时检测出pX330-xCas9 T1、T2和T3的基因靶向能力。最后一个sgRNA载体显示最高的检测信号，随后用于绵羊基因编辑研究。按照此协议，可以进行 T7EI 测定或核糖核酸酶测定。这些额外的措施给内源基因编辑更准确的印象。

Research

JoVE Journal

Bioengineering

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

使用光声血流循环黑色素瘤细胞的检测和隔离

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科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

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使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

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In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...