Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9 based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated knock-down AGO1 cell lines. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

中文翻译：

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一种基于质粒的选择性系统，可产生CRISPR / Cas9基因编辑和敲入的蚊子细胞系

伊蚊（ae）埃及和埃及。白纹伊蚊蚊子传播虫媒世界各地的疾病，引起〜每年70万例死亡。遗传突变体是询问基本的媒介生物学和对病毒感染重要的蚊子宿主因子的有价值的工具。但是，与模型生物相比，在蚊子中很少描述遗传突变体。应用基于CRISPR / Cas9的基因编辑相对容易，已经改变了基因组工程，并迅速增加了蚊子中可用基因突变体的数量。然而，体内研究对于筛选大量突变体或缺乏昆虫的实验室可能不切实际。因此，使CRISPR / Cas9系统适应常见的蚊子细胞系将是有用的。在这项研究中，我们产生和其特征在于优化的蚊子，基于质粒CRISPR / Cas9系统用于U4.4（白纹伊蚊）和Aag2（埃及伊蚊）细胞系。我们展示了AGO1基因座和分离的敲低AGO1细胞系的高效编辑。此外，我们使用同源性指导的修复方法，在内源性AGO1的N端建立了具有3xFLAG标签的敲入Aag2细胞系。这些经过实验验证的质粒是通用的，具有成本效益的，并且可以有效编辑在虫媒病毒研究中广泛使用的具有免疫活性的蚊子细胞系。