Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.

Qiang Yan, Yingying He, Yang Yue, Luyao Jie, Tingmei Wen, Yumiao Zhao, Min Zhang, Tingting Zhang
{"title":"Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.","authors":"Qiang Yan, Yingying He, Yang Yue, Luyao Jie, Tingmei Wen, Yumiao Zhao, Min Zhang, Tingting Zhang","doi":"10.3791/63629","DOIUrl":null,"url":null,"abstract":"<p><p>The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.</p>","PeriodicalId":90591,"journal":{"name":"Journal of electroanalytical chemistry and interfacial electrochemistry","volume":"300 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2022-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of electroanalytical chemistry and interfacial electrochemistry","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/63629","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

利用 CRISPR/Cas9 技术构建迁徙蝗虫的同源突变体。
迁飞蝗虫(Locusta migratoria)不仅是给人类造成巨大经济损失的世界性瘟疫蝗虫之一,也是昆虫变态的重要研究模型。CRISPR/Cas9系统可以准确定位特定的DNA位点,并在目标位点内进行裂解,高效地引入双链断裂,诱导目标基因敲除或将新的基因片段整合到特定位点。CRISPR/Cas9 介导的基因组编辑是解决蝗虫研究中遇到的问题的有力工具,也是一种很有前景的蝗虫控制技术。本研究提供了一套系统的方案,利用Cas9蛋白和单导RNA(sgRNA)复合物在迁徙蝗虫中进行CRISPR/Cas9介导的基因敲除。详细描述了目标位点的选择和 sgRNA 的设计,随后是 sgRNA 的体外合成和验证。随后的程序包括卵筏收集和鞣卵分离,以实现低死亡率的成功显微注射、卵培养、突变率的初步估计、蝗虫繁殖以及突变体的检测、保存和通过,以确保编辑蝗虫种群的稳定性。该方法可作为基于 CRISPR/Cas9 的基因编辑技术在迁徙蝗虫和其他昆虫中应用的参考。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信