CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, Aedes aegypti, a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, kynurenine 3-monooxygenase (kmo), and the juvenile hormone receptor, Methoprene-tolerant (Met). PiggyBac transformation vectors were prepared to express sgRNAs targeting kmo and Met under the control of the U6 promoter. Transgenic Ae. aegypti expressing kmo-sgRNA or Met-sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in kmo-sgRNA and Met-sgRNA groups, respectively, showed mutations in their target genes.

中文翻译：

---

扩展疾病媒介埃及伊蚊基因组编辑工具包：表达 Cas9 和单向导 RNA 的转基因株系诱导高效诱变

CRISPR-Cas9 介导的基因组编辑方法正用于基因功能分析。然而，很难确定基因敲除突变体的基因敲除不会导致不同的表型。为了克服疾病载体中的这个问题，埃及伊蚊，一种转基因 Cas9/单向导 RNA (sgRNA) 方法，被用于敲除眼睛标记基因犬尿氨酸 3-单加氧酶( kmo ) 和保幼激素受体Methoprene-宽容（满足）。制备 PiggyBac 转化载体以在 U6 启动子的控制下表达靶向kmo和Met的 sgRNA。转基因Ae。表达kmo 的埃及伊蚊-sgRNA 或Met -sgRNA 在 U6 启动子控制下产生，增强型绿色荧光蛋白 (eGFP) 在 hr5ie1 启动子控制下产生。U6-sgRNA 成虫与 AAEL010097-Cas9 成虫交配。筛选后代，选择表达 eGFP 和 DsRed 的昆虫并评估目标基因的突变。kmo -sgRNA 和Met -sgRNA 组中 eGFP 和 DsRed 均呈阳性的后代中，分别约有 77% 和 78%在其靶基因中显示出突变。