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
Summary
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。
Abstract
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）。
Introduction
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）。
Protocol
1. sgRNA寡核苷酸设计 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。 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….
Representative Results
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（?…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| A new generation of full touch screen gradient PCR instrument | LongGene | A200 | Target gene amplification |
| AscI restriction enzymes | New England Biolabs | R0558V | Cutting target vectors |
| BbsI restriction enzyme | New England Biolabs | R0539S | Cutting target vectors |
| Clean workbench | AIRTECH | SW-CJ-2FD/VS-1300L-U | A partial purification device in the form of a vertical laminar flow, which creates a local high clean air environment |
| DH5α Competent Cells | TaKaRa | K613 | Plasmid vector transformation |
| Dual-Luciferas Reporter Assay System | Promega | E1910 | Dual-luciferas reporter assay |
| Electric thermostatic water bath | Sanfa Scientific Instruments | DK-S24 | Heating reagent by constant temperature in water bath |
| Electrophoresis | Beijing Liuyi Biotechnology Co., Ltd. | DYY-6C | Control voltage, current, etc. |
| Eppendorf Reference 2 | Eppendorf China Ltd. | Reference 2 | Accurately draw and transfer traces of liquid |
| Gel imaging analyzer | Beijing Liuyi Biotechnology Co., Ltd. | WD-9413B | For the analysis of electrophoresis gel images |
| GloMax 20/20 Luminometer | Promega | E5311 | Detect dual luciferase activity |
| High speed refrigerated centrifuge | BMH | sigma 3K15 | Nucleic acid extraction and purification |
| Intelligent biochemical incubator | Sanfa Scientific Instruments | SHP-160 | Provide a suitable temperature environment for the enzyme digestion experiment |
| LB Broth Agar | Sangon Biotech | A507003-0250 | For the cultivation of E.coli |
| Lipofectamine 3000 Transfection Reagent Kit | Thermo Fisher | L3000015 | DNA Transfection |
| SalI restriction enzymes | New England Biolabs | R3138V | Cutting target vectors |
| SanPrep Column DNA Gel Extraction Kit | Sangon Biotech | B518131-0050 | Recycling DNA fragments |
| SanPrep Column Plasmid Mini-Preps Kit | Sangon Biotech | B518191-0100 | Extraction of plasmid DNA |
| T4 DNA Ligase | New England Biolabs | M0202V | Link DNA fragment |
| TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 | TaKaRa | 9761 | DNA purification |
| Vertical pressure steam sterilizer | JIBIMED | LS-50LD | High temperature and autoclave to kill bacteria, fungi and other microorganisms in laboratory equipment |
| Water bath thermostat | Changzhou Guoyu Instrument Manufacturing Co., Ltd. | SHZ-82 | Let the bacteria keep shaking, which is good for contact with air. |
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Citazione di questo articolo
Li, H., Qin, H., Zhang, N., Zhao, J., Xin, J., Perez-Campo, F. M., Liu, H. Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System. J. Vis. Exp. (166), e59639, doi:10.3791/59639 (2020).