Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats‐associated protein 9 (CRISPR‐Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR‐Cas9‐targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.

中文翻译：

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使用簇状规则间隔的短回文重复序列相关蛋白9在疾病载体库克斯库克斯库蚊中产生可遗传种系突变的方法。

库克斯库蚊（Culex quinquefasciatus）是许多对人类和动物健康产生不利影响的疾病的媒介。但是，与其他蚊子载体相比，有限的基因组工程技术已被表征为这种载体。基于聚类的规则间隔的短回文重复序列相关蛋白9（CRISPR-Cas9）技术是进行基因组工程和功能遗传学的强大工具，因此已改变了许多生物的遗传研究。我们的目标是改进西洋参（C. quinquefasciatus）基因组编辑的有限技术在该载体中创建针对CRISPR-Cas9的诱变的可再现且直接的方法。在这里，我们描述了实现高胚胎存活率和诱变率的方法，并提供了有关注射用品和程序，胚胎处理和指导RNA（gRNA）目标设计的详细信息。通过这些努力，我们实现了胚胎存活率和种系诱变率，大大超过了该载体先前报道的率。该作品也是第一个表征白色的作品该物种中的基因标记，是该载体未来转基因或诱变的有价值的表型标记。总体而言，这些工具为该重要疾病载体的未来功能遗传研究提供了框架，并可能支持未来可用于控制该载体的基因驱动和遗传技术的发展。