Department of Zoology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Department of Zoology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
Cold Spring Harb Protoc. 2024 Sep 3;2024(9):pdb.prot108340. doi: 10.1101/pdb.prot108340.
Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.
创建转基因蚊子可以进行基础蚊子生物学的机制研究和新型媒介控制策略的开发。CRISPR-Cas9 基因编辑技术已经彻底改变了基因编辑，包括在蚊子中。本方案详细介绍了通过 CRISPR-Cas9 对蚊子进行基因编辑的部分过程，包括测试和验证单指导 RNA（sgRNA）。基因编辑活性取决于所用 sgRNA 的序列，因此在大规模生成突变体或转基因之前，应该对 sgRNA 活性进行验证。sgRNA 使用在线工具设计，并在<1 小时内合成。通过胚胎显微注射生成突变体或转基因后，通过快速基因型聚合酶链反应（PCR）和 DNA 测序验证 sgRNA 活性。
