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 into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. 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 Cx. 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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CRISPR 基因敲除评估作为蚊子库蚊基因工程的潜在目标

Culex quinquefasciatus蚊子是几种人类和动物病原体的全球广泛传播媒介。他们的生物学和行为使他们能够在靠近城市地区茁壮成长，使他们成为持续的公共卫生威胁。它们混合的鸟类/哺乳动物摄食行为进一步为人畜共患病病原体传播给人类提供了一种载体，并且分别对岛屿鸟类的保护构成了威胁。CRISPR的出现导致了野生蚊子种群基因工程新技术的发展。然而，对Cx 的研究。与其他疾病载体相比，quinquefasciatus一直落后。在这里，我们使用这个工具来破坏Cx 中的一组五个色素沉着基因。五花肉当改变时，会导致可见的、纯合可行的表型。我们在不同的实验室和两种不同的Cx菌株中进一步验证了这种方法。与潜在的未来公共卫生和鸟类保护应用相关的quinquefasciatus。我们生成了一个双突变系，证明了在一个个体中顺序组合多个此类突变的可能性。最后，我们的目标两个位点，doublesex在性别决定途径和proboscipedia，一个HOX基因，这表明这些方法应用到新的目标的灵活性。我们的工作提供了一个由七个经过验证的基因座组成的平台，可用于Cx 中的靶向诱变。五花肉以及该物种遗传抑制策略的未来发展。此外，这里产生的突变系可能对使用这种模式生物的研究界具有广泛的用途，因为它们可以用作转基因递送的靶标，其中被破坏的基因的副本可以作为易于评分的转基因标记包括在内。