CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定
1School of Chemical and Environmental Engineering,China University of Mining and Technology, 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,Chinese Academy of Agricultural Sciences, 3Guangdong Laboratory of Lingnan Modern Agriculture, Shenzhen; Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences
Summary
这里展示的是 Helicoverpa armigera （Hübner）胚胎显微注射和通过CRISPR / Cas9基因组编辑创建的敲除突变体鉴定的方案。突变昆虫能够进一步研究体内不同基因的功能和相互作用。
Abstract
棉铃虫， Helicoverpa armigera，是世界上最具破坏性的害虫之一。分子遗传学，生理学，功能基因组学和行为研究的结合使 H. armigera 成为鳞翅目夜蛾科的模型物种。为了研究不同基因的体内功能和相互作用，簇状规则间隔的短回文重复序列（CRISPR）/相关蛋白9（Cas9）基因组编辑技术是一种用于进行功能基因组研究的方便有效的方法。在这项研究中，我们提供了一种循序渐进的系统方法，使用CRISPR / Cas9系统完成 臂状芽胞 杆菌中的基因敲除。详细描述了引导RNA（gRNA）的设计和合成。然后，总结了由引导RNA（gRNA）创建，胚胎收集，显微注射，昆虫饲养和突变体检测的基因特异性引物设计组成的后续步骤。最后，提供故障排除建议和注释，以提高基因编辑的效率。我们的方法将作为CRISPR / Cas9基因组编辑在 臂状芽孢 杆菌以及其他鳞翅目蛾中的应用的参考。
Introduction
基因组编辑技术的应用为实现不同物种的靶基因突变体提供了有效的工具。簇状规则间隔短回文重复序列（CRISPR）/相关蛋白9（Cas9）系统的出现提供了一种操纵基因组的新方法1。CRISPR/Cas9系统由引导RNA（gRNA）和Cas9内切酶2，3组成，而gRNA可以进一步分为两部分，靶向互补CRISPR RNA（crRNA）和反式激活crRNA（tracrRNA）。gRNA与Cas9内切酶整合并形成核糖核蛋白（RNP）。使用gRNA，Cas9内切酶可以通过碱基互补被引导到基因组的特定位点。Cas9的RuvC和HNH结构域在原始间隔体相邻基序（PAM）序列之前切割基因组的三个碱基的靶位点，并产生双链断裂（DSB）。然后，DNA切割可以通过两种机制进行修复，即非同源末端连接（NHEJ）或同源定向修复（HDR）4。DSB的修复引入了插入或缺失作为灭活靶向基因的方法，可能导致基因功能完全丧失。因此，CRISPR/Cas9系统的可异性和特异性使其成为表征体内基因功能和分析基因相互作用的可靠方法5。
CRISPR/Cas9系统具有诸多优点，已应用于生物医学6、7、基因治疗8、9、农业等各个领域10、11、12，并已用于各种生物系统，包括微生物13、植物14、15、线虫16 和哺乳动物17.在无脊椎动物中，许多昆虫物种都经过CRISPR/Cas9基因组编辑，如果蝇 果蝇黑腹果 蝇等18，19，20，21，22。
Helicoverpa armigera是全球最具破坏性的害虫之一23，并损害了许多作物，包括棉花，大豆和高粱24，25。随着测序技术的发展，阿米格拉氏菌的基因组以及一系列鳞翅目昆虫物种的基因组已经完全测序26，27，28，29。近年来，从这些昆虫中鉴定和表征了大量的抗性和嗅觉受体基因19，27，28，29。在臂状螺旋体中已经发现了一些与抗性相关的基因，例如编码钙粘蛋白30，ATP结合盒转运蛋白31，32以及HaTSPAN133的基因。使用CRISPR / Cas9技术敲除这些基因可导致对易感菌株中苏云金芽孢杆菌（BT）毒素的高度抗性。此外，Chang等人（2017）敲除了一种信息素受体，这验证了其在交配时间调节方面的重要功能19。这些报告表明，CRISPR/Cas9可以作为研究昆虫系统中体内基因功能的有效工具。然而，CRISPR/Cas9修饰在昆虫系统中的详细程序仍不完整，这限制了其在昆虫功能基因组学中的应用范围。
在这里，我们提出了一种使用CRISPR / Cas9系统敲除 臂状 芽蒿蒿中功能基因的方案。提供了详细的分步方案，包括设计和制备用于gRNA生产，胚胎收集，显微注射，昆虫饲养和突变体鉴定的基因特异性引物。该协议可作为操纵 臂状蛊 虫中任何功能基因的宝贵参考，并且可以扩展到其他鳞翅目物种。
Protocol
1. 基因特异性引物的设计及sgRNA的制备 通过PCR扩增和测序分析验证目标基因中的保守基因组区域。从 臂状 芽胞杆菌的基因组DNA中扩增靶基因，并区分外显子和内含子。注意：引导位点的序列特异性对于避免脱靶基因编辑是必要的。在外显子中搜索可能的引导位点接近基因的5’UTR。然后，重要的是要确保基因完全无功能。用于制备sgRNA的流路摘要如图 1所示。…
Representative Results
该协议提供了使用CRISPR / Cas9技术获得 臂状芽蒿 的基因敲除系的详细步骤。总结了通过该协议获得的代表性结果，用于gDNA选择，胚胎收集和注射，昆虫饲养和突变体检测。 在这项研究中，我们感兴趣的基因的靶位点位于其第二个外显子中（图2A）。该位点高度保守，使用琼脂糖凝胶电泳确认合成sg…
Discussion
CRISPR/Cas9系统的应用为分析基因功能和基因间相互作用提供了有力的技术支持。我们在这里介绍的详细方案证明了通过CRISPR / Cas9基因组编辑在 H. armigera 中产生纯合子突变体。这种可靠的程序为 臂状芽孢杆菌的定向基因诱变提供了一种直接的方法。
CRISPR靶点的选择可能会影响诱变效率37.在该协议中，我们比较并分析了来自在线网站CRISOR的多个结…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（31725023，31861133019 GW，31171912 CY）的支持。
Materials
| 2kb DNA ladder | TransGen Biotech | BM101 | |
| Capillary Glass | World Precision Instrucments | 504949 | referred to as "capillary glass" in the protocol |
| Double Sided Tape | Minnesota Mining and Manufacturing Corporation | 665 | |
| Eppendorf FemtoJet 4i Microinjector | Eppendorf Corporate | E5252000021 | |
| Eppendorf InjectMan 4 micromanipulator | Eppendorf Corporate | 5192000051 | |
| Eppendorf Microloader Pipette Tips | Eppendorf Corporate | G2835241 | |
| GeneArt Precision gRNA Synthesis Kit | Thermo Fisher Scientific | A29377 | |
| Microscope Slide | Sail Brand | 7105 | |
| Olympus Microscope | Olympus Corporation | SZX16 | |
| PrimeSTAR HS (Premix) | Takara Biomedical Technology | R040 | used for mutant detection |
| Sutter Micropipette Puller | Sutter Instrument Company | P-1000 | |
| TIANamp Genomic DNA Kit | TIANGEN Corporate | DP304-03 | |
| TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | A36499 |
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Cite This Article
Ai, D., Wang, B., Fan, Z., Fu, Y., Yu, C., Wang, G. Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner). J. Vis. Exp. (173), e62068, doi:10.3791/62068 (2021).