Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex quinquefasciatus has been lagging compared to other disease vectors. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Culex quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.

中文翻译：

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通过CRISPR评估色素沉着基因敲除作为库蚊（Culex quinquefasciatus）基因工程的潜在目标

昆蚊（Culex quinquefasciatus）蚊子是几种人类和动物病原体的全球传播媒介。他们的生物学和行为使他们能够在靠近市区的地方，壮成长，使之成为持续不断的公共卫生威胁。它们的鸟类/哺乳动物混合喂养行为进一步为人畜共患病原体向人的传播提供了一种媒介，并且分别对保护岛屿鸟类构成了威胁。CRISPR的出现导致了野生蚊子种群基因工程的新技术的发展，但是与其他疾病载体相比，昆西库蚊（Culex quinquefasciatus）的研究一直滞后。在这里，我们使用该工具破坏了库克斯库克斯库克斯犬的五个色素沉着基因的集合，当这些色素沉着基因被改变时，它们会导致可见的，纯合的有生命力的表型​​。我们进一步在单独的实验室和与潜在的未来公共卫生和鸟类保护应用有关的两种不同的库克斯库克斯菌中验证了这种方法。我们生成了一个双突变线，表明了在单个个体中顺序组合多个此类突变的可能性。最后，我们针对两个基因座，即性别决定途径中的双性恋和probscipedia a hox基因，证明了这些方法适用于新型靶标的灵活性。我们的工作提供了一个包含七个经过验证的基因座的平台，该平台可用于昆西库蚊的定向诱变以及该物种的遗传抑制策略的未来发展。此外，此处产生的突变体系可能会使用这种模式生物对研究界产生广泛的应用，