Germline Transformation of Ticks

蜱的种系转化

• 批准号: 10737473
• 负责人: Monika Gulia-Nuss
• 金额: $ 68.63万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-11 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737473
• 关键词: Agriculture; Anterior; Arthropod Vectors; Arthropods; Biology; Black-legged Tick; Body Weight; CRISPR/Cas technology; Cattle; Cell Line; Cell Nucleus; Cell Proliferation; Cells; Chimeric Proteins; Complementary RNA; Culicidae; DNA; DNA Ligation; DNA Repair; Data; Development; Disease; Disease Vectors; Docking; Double Strand Break Repair; Drosophila genus; Economics; Embryo; Embryonic Development; Event; Excision; Exposure to; Frequencies; Future; Gene Expression; Generations; Genes; Genetic; Genetic Transformation; Genome; Genome engineering; Germ; Germ Cells; Germ-Line Mutation; Goals; Grant; Guide RNA; Heritability; Incidence; Injections; Insecta; Knock-in; Knock-out; Knowledge; Location; Lyme Disease; Mammalian Cell; Mediating; Methods; Modification; Molecular; Molecular Biology; Mutation; Nonhomologous DNA End Joining; Nucleotides; Organism; Outcome; Pathway interactions; Phenotype; Play; Population; Protocols documentation; Public Health; RNA interference screen; Research; Scheme; Site; Somatic Cell; Structure of primordial sex cell; System; Techniques; Testing; Tick Control; Tick-Borne Diseases; Ticks; Time; Transgenes; Transgenic Organisms; Treatment Cost; United States; Vertebrates; Work; cost; disorder control; egg; endonuclease; experimental study; functional genomics; gene function; genetic information; genome editing; improved; interest; knockout gene; migration; milk production; model organism; nano; new therapeutic target; novel strategies; novel vaccines; pathogen; precise genome editing; prevent; promoter; repaired; single-cell RNA sequencing; tool; transmission process; vector

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.

项目摘要 蜱及其传播的病原体给全世界的公共卫生和农业带来了巨大的成本。 例如，肩胛硬蜱，莱姆病（LD）在美国的主要载体，是负责 每年超过30万例LD病例。微小牛蜱（Rhiphicephalus（Boophilus）microplus）造成的经济损失 双重：牛的体重和产奶量降低，以及用于预防的治疗费用 疾病和控制蜱。蜱和蜱传疾病的发病率和分布增加， 更好地了解病媒生物学，以开发控制蜱虫的新方法。遗传学的最新进展 转基因技术，特别是CRISPR/Cas9系统极大地促进了功能基因组学研究。 这些进展现在允许阐明非模式生物（如蜱）中的基因功能。但是，在这方面， 由于蜱虫独特的生物学特性，一些技术障碍阻碍了基因编辑的实现。 应用于研究蜱虫分子生物学，最明显的是缺乏胚胎注射方案和对 早期的胚胎活动。我们通过R21赠款克服了重大障碍， 胚胎注射方案和第一个原理验证的蜱基因敲除。然而，没有遗传插入 已经在蜱虫中观察到了。在正确的时间注射卵子是至关重要的，这样引入的材料可以 进入未来生殖细胞的细胞核（在细胞化之前）并产生稳定的生殖系转化体。 CRISPR/Cas9使用与靶DNA互补的引导RNA，并通过靶向RNA引导DNA切割。 Cas9内切核酸酶。基因组序列的修饰发生在双链断裂（DSB）修复期间， 而起作用的分子途径决定了序列变化的类型。Canonical 非同源末端连接（cNHEJ）和替代末端连接途径，例如微同源介导的 末端连接（MMEJ）通过DNA末端的连接进行，并导致靶向但不精确的编辑（通常较小 插入或缺失）导致基因敲除。然而，两个或更多个核苷酸的微同源性 在通过切除进行DNA切割后暴露的DNA片段可用于在MMEJ修复期间进行精确编辑。 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，蜱虫经常使用MMEJ途径进行DSB修复。在这份提案中，我们将填补 我们的知识差距，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，以更好地了解基因表达过程中， 早期胚胎发育（Aim 1）。然后，我们将利用我们以前的发现来生成生殖系编辑的 通过开发Vasa-Cas9系进行有效和可访问的敲除研究。我们将比较 MMEJ和HDR用于敲入实验以整合转基因的效率（目的2）。预期成果 我们的工作将提供新的工具，以确定许多蜱表型的遗传基础，包括那些 参与病原体的传播