东方果蝇背侧芽孢杆菌CRISPR/Cas9诱变实验方案
1Key Laboratory of Sustainable Forest Ecosystem Management – Ministry of Education,Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences
Summary
本文介绍了东方果蝇背蝇CRISPR/Cas9诱变的分步方案。该标准化协议提供的详细步骤将作为产生突变果蝇的有用指南，用于背双歧杆菌的功能基因研究。
Abstract
东方果蝇 Bactrocera dorsalis 是一种高度侵入性和适应性的害虫物种，对柑橘和全球150多种其他水果作物造成损害。由于成年果蝇具有很强的飞行能力，雌性果蝇在果皮下产卵，因此需要直接接触害虫的杀虫剂通常在田间表现不佳。随着分子生物学工具和高通量测序技术的发展，许多科学家正在尝试制定环保的害虫管理策略。这些包括基于RNAi或基因编辑的杀虫剂，它们下调或沉默各种害虫中的基因（分子靶标），例如参与搜索行为的嗅觉基因。为了使这些策略适用于东方果蝇控制，需要有效的功能基因研究方法。在 背苜蓿 的存活和繁殖中具有关键功能的基因是基因敲低和/或沉默的良好分子靶标。CRISPR/Cas9系统是一种用于基因编辑的可靠技术，特别是在昆虫中。本文提出了一种系统 的方法，包括引导RNA的设计和合成、胚胎采集、胚胎注射、昆虫饲养和突变体筛选。这些协议将作为对背 侧双歧杆菌功能基因研究感兴趣的研究人员产生突变果蝇的有用指南。
Introduction
东方果蝇 Bactrocera dorsalis ，是一种世界性的害虫物种，对 150 多种水果作物造成损害，包括番石榴、芒果、尤金属、苏里南樱桃、柑橘、枇杷和木瓜1。仅在广东省（中国）造成的损失估计就超过2亿元人民币。成年雌性将卵插入成熟或成熟果实的皮下，导致果实腐烂和脱落，从而降低果实品质和作物总产量2。由于成年果蝇具有很强的飞行能力，它们的幼虫钻入果皮，因此需要直接接触害虫的杀虫剂在田间表现不佳。此外，杀虫剂的广泛使用增加了 背双歧杆菌 对各种农业化学品的抵抗力，使得控制这些有害害虫变得更加困难3。因此，迫切需要制定有效和环保的害虫管理策略。
最近，随着分子生物学工具和高通量测序技术的发展，科学家们正试图开发环境友好的害虫管理策略，例如RNAi，该策略针对各种害虫的重要基因（分子靶标）的功能。通过功能基因研究，可以鉴定出对害虫生存和繁殖至关重要的基因，并进一步作为改进专门针对性和环境友好型害虫管理工具的潜在分子靶标4。为了使这些策略适应东方果蝇控制，需要有效的功能基因研究方法。
CRISPR/Cas（成簇规则间隔短回文重复序列/CRISPR相关）核酸内切酶系统最初是在细菌和古细菌中发现的，并发现是一种参与识别和降解外来细胞内DNA的适应性机制，例如通过感染噬菌体引入的机制5。在II型CRISPR系统中，Cas9核酸内切酶在小相关RNA（crRNA和tracrRNA）的引导下切割侵入DNA6，7，8，并已成为迄今为止使用最广泛的基因编辑工具之一9，10，11，12。由于CRISPR/Cas9系统具有基因沉默效率高、成本低等优点，因此已被应用于各种昆虫物种的基因编辑，包括埃及伊蚊13，14、蝗虫迁徙15和森16。在B. dorsalis中，与体色，翅膀二态性和性别决定相关的基因已经使用CRISPR / Cas917，18，19成功敲除。然而，CRISPR / Cas9在这种昆虫中应用的详细程序仍然不完整。此外，研究人员为背侧双歧杆菌基因编辑提供的一些步骤也各不相同，需要标准化。例如，Cas9的形式在已发表的参考文献17，18，19中是不同的。
本文提供了利用CRISPR/Cas9系统诱变背 苜蓿 的系统方法，包括引导RNA的设计和合成、胚胎采集、胚胎注射、昆虫饲养和突变体筛选。该协议将作为对 背侧双歧杆菌功能基因研究感兴趣的研究人员产生突变苍蝇的有用指南。
Protocol
1. 靶标设计与sgRNA的 体外 合成 通过背侧双歧杆菌基因组的生物信息学分析，预测感兴趣的靶基因的结构并确定外显子和内含子之间的边界（此处使用的软件应用程序列在材料表中）。注意：BLAT20 用于搜索基因组中潜在的基因位点。使用Hisat221将高质量的RNA-seq读数（转录组）与获得的基因位点对齐。Samtoo…
Representative Results
该协议提供了使用CRISPR / Cas9技术开发背 侧双歧杆菌 突变体的详细步骤，包括gDNA选择，收集胚胎和显微注射，昆虫维持和突变体筛选的代表性结果。 所选基因的靶位点的示例位于第三个外显子中（图1C）。该位点高度保守，通过凝胶电泳检测合成gRNA的DNA模板（图1D）和体 外 转录获得的gRNA的单条带（?…
Discussion
CRISPR/Cas9系统是使用最广泛的基因编辑工具，具有多种应用，如基因Threpy30，作物育种31和基因功能的基础研究32。该系统已被应用于各种昆虫物种的基因编辑，并已成为害虫功能基因研究的有效工具。我们在这里介绍的方案标准化了引导RNA的设计和合成，收集胚胎，胚胎注射，昆虫饲养和突变体筛选的过程。此外，还增加了一个故障排除表来?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了深圳市科技计划（批准号：KQTD20180411143628272）和深圳市大鹏新区科技创新与产业发展专项资金（批准号：PT202101-02）的支持。
Materials
| 6x DNA Loading Buffer | TransGen Biotech | GH101-01 | |
| Artificial climate chamber | ShangHai BluePard | MGC-350P | |
| AxyPrep Genomic DNA Mini-Extraction Kit | Axygen | AP-MN-MS-GDNA-250G | |
| BLAT | NA | NA | For searching potential gene loci in the genome |
| Capillary Glass | WPI | 1B100F-4 | |
| Eppendorf InjectMan 4 micromanipulator | Eppendorf | InjectMan 4 | |
| GeneArt Precision gRNA Synthesis Kit | Thermo Fisher Scientific | A29377 | |
| Hisat2 | NA | NA | For aligning the transcriptome to the acquired gene loci |
| IGV | NA | NA | For visualizing the results from Transdecoder |
| Microgrinder | NARISHIGE | EG-401 | |
| Olympus Microscope | Olympus Corporation | SZ2-ILST | |
| pEASY-Blunt Cloning Kit | TransGen Biotech | CB101-02 | https://www.transgenbiotech.com/data/upload/pdf/CB101_2022-07-14.pdf |
| Phenol red solution | Sigma-Aldrich | P0290-100ML | |
| Pipette cookbook 2018 P-97 & P-1000 Micropipette Pullers | Instrument Company | https://www.sutter.com/PDFs/cookbook.pdf | |
| PrimeSTAR HS (Premix) | Takara Biomedical Technology | R040A | |
| SAMtools | NA | NA | For generating the sorted bam files |
| sgRNAcas9-AI | NA | NA | sgRNA design http://123.57.239.141:8080/home |
| Sutter Micropipette Puller Sutter | Instrument Company | P-97 | |
| Trans2K DNA Marker | TransGen Biotech | BM101-02 | |
| Transdecoder | NA | NA | For combining the results of assemble transcripts and gene loci information https://github.com/TransDecoder/TransDecoder/releases/tag/TransDecoder-v5.5.0 |
| TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | A36498 | |
| Ultra-trace biological detector | Thermo Fisher Scientific | Nanodrop 2000C |
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Cite This Article
Yuan, J., Zhang, J., Zhang, Y., QiQiGe, W., Liu, W., Yan, S., Wang, G. Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis. J. Vis. Exp. (187), e64195, doi:10.3791/64195 (2022).