Germline Transformation of Ticks

点击翻译按钮获取中文摘要

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.