Germline Transformation of Ticks

项目总结 虱子及其传播的病原体给世界各地的公共卫生和农业带来了巨大的成本。 例如，在美国传播莱姆病(LD)的主要媒介--肩部硬蜱 每年超过300,000个LD案件。微增型Rhiphicephalus的经济损失为 两倍：减少牛的体重和产奶量，并减少用于预防的治疗费用 疾病和控制扁虱。壁虱和壁虱传播疾病的发病率和分布的增加需要 更好地了解媒介生物学，以开发新的滴虫控制方法。遗传学的最新进展 转型技术，特别是CRISPR/Cas9系统极大地促进了功能基因组学的研究。 这些进展现在可以阐明非模式生物，如扁虱的基因功能。然而， 由于扁虱独特的生物学特性，有几个技术障碍阻碍了基因编辑 应用于扁虱分子生物学的研究，最显著的是缺乏胚胎注射方案和对 早期的胚胎事件。我们通过我们的R21赠款克服了重大障碍，通过开发 胚胎注射方案和第一个原理证明的tick基因敲除。但是，没有可遗传的插入 已经在扁虱身上观察到了。在正确的时间注射卵子是至关重要的，这样引入的材料就可以 获取未来生殖细胞的细胞核(在细胞化之前)，并创造稳定的生殖系转化体。 CRISPR/Cas9使用与靶DNA互补的引导RNA，并通过 Cas9内切酶。基因组序列的修改发生在双链断裂(DSB)修复期间， 而发挥作用的分子途径决定了序列变化的类型。典范 非同源末端连接(CNHEJ)和替代末端连接途径，如微同源介导的 末端连接(MMEJ)通过连接DNA末端进行，并导致定向但不精确的编辑(通常较小 插入或缺失)导致基因敲除。然而，两个或更多核苷酸的微同源性 切除DNA裂解后暴露的DNA可用于MMEJ修复过程中的精确编辑。 同源定向修复(HDR)使用支持精确基因组编辑的外源DNA修复模板。 我们以前的工作表明，扁虱经常使用MMEJ途径进行DSB修复。在这份提案中，我们将填充 我们的知识差距是通过首先了解原始生殖细胞形成的时间和地点 标记以及利用单细胞RNAseq技术更好地了解基因在 早期胚胎发育(目标1)。然后我们将利用我们之前的发现来生成经过种系编辑的 通过开发VASA-Cas9品系进行高效和可访问的基因敲除研究。我们将比较 MMEJ和HDR对整合转基因的敲入实验的效率(目标2)。预期结果 我们的工作将提供新的工具来确定许多扁虱表型的遗传基础，包括那些 参与了病原体的传播。

PROJECT SUMMARY Ticks and the pathogens they transmit incur significant costs to public health and agriculture worldwide. For instance, Ixodes scapularis, the primary vector of Lyme disease (LD) in the United States, is responsible for over 300,000 LD cases annually. The economic losses due to Rhiphicephalus (Boophilus) microplus are two-fold: reduced body weight and milk production in cattle and the treatment cost employed to prevent disease and control ticks. Increased incidence and distribution of ticks and tick-borne diseases necessitates a better understanding of vector biology to develop new approaches for tick control. Recent advances in genetic transformation techniques, esp. CRISPR/Cas9 system has immensely facilitated functional genomics studies. These advances now allow the elucidation of gene functions in non-model organisms such as ticks. However, because of the unique biology of ticks, several technical hurdles have prevented gene-editing from being applied to study tick molecular biology, most notably lack of an embryo injection protocol and understanding of the early embryonic events. We overcame significant impediments through our R21 grant by developing embryo injection protocols and the first proof-of-principle tick gene knockout. However, no heritable insertions have been observed in ticks yet. It is essential to inject eggs at the right time so that introduced material can access the nuclei of the future germ cells (before cellularization) and create stable germline transformants. CRISPR/Cas9 uses a guide RNA complementary to the target DNA and directs DNA cleavage by the Cas9 endonuclease. Modification of the genome sequence occurs during double-stranded break (DSB) repair, and the molecular pathways that come into play determine the type of sequence change. Canonical nonhomologous end-joining (cNHEJ) and alternative end-joining pathways such as micro-homology-mediated end-joining (MMEJ) proceed by ligation of DNA ends and result in targeted but imprecise edits (generally small insertions or deletions) resulting in gene knockout. However, microhomologies of two or more nucleotides exposed after DNA cleavage through resection could be used for precise editing during repair by MMEJ. Homology-directed repair (HDR) uses an exogenous DNA repair template that supports precise genome editing. Our previous work suggests that ticks frequently use MMEJ pathways for DSB repair. In this proposal, we will fill our knowledge gaps by first understanding the timing and site of primordial germ cell formation using known markers as well as utilizing the single-cell RNAseq technique to better understand the gene expression during early embryonic development (Aim 1). We will then leverage our previous findings to generate germline-edited ticks by developing Vasa-Cas9 lines for efficient and accessible knockout studies. We will compare the efficiencies of MMEJ and HDR for knock-in experiments to integrate transgenes (Aim 2). The expected outcomes of our work will provide new tools to determine the genetic basis of many tick phenotypes, including those involved in pathogen transmission.