Development of CRISPR/Cas system for study of trans-sialidase family genes in T. cruzi

 描述（由申请人提供）：克氏锥虫基因组的复杂性和独特特征以及操纵该基因组的工具的相对缺乏是导致恰加斯病的寄生虫持久性和致病性研究中的一些挑战。作为美洲影响最大的传染病，有多达2000万人和无数动物受到影响，T。cruzi的问题是诊断工具差，治疗选择不足，没有有效的疫苗。通过改进T的基因组编辑工具，将大大促进所有这些领域的新发现。克鲁兹我们最近将CRISPR技术应用于T. Cruzi和Cruzi证明了显著增加的基因敲除和基因插入效率，为这种重要的人类病原体的遗传分析提供了无与伦比的机会。在这个项目中，我们将进一步增强T. cruzi的研究，通过提高调节Cas9表达的能力，更好地定义用于修复Cas9介导的双链断裂的DNA重组机制，并使用该信息优化T.克鲁兹使用CRISPR-Cas9所展示的多重能力，我们将评估编码转唾液酸酶（ts）分子的大量基因突变的能力，以评估ts分子家族对T.克鲁兹该建议结合了新的使用和进一步发展的一个强大的遗传系统，我们证明是非常有效的T。cruzi，带着一个重要的生物学问题，到目前为止，这个问题还不是实验上可以解决的。了解大基因家族在T.如果我们能够减少家庭成员的数量并观察其后果，cruzi才有可能。这项工作的完成也有望将CRISPR-Cas系统牢固地确立为T.克鲁兹

 DESCRIPTION (provided by applicant): The complexity and unique characteristics of the Trypanosoma cruzi genome and the relative paucity of tools to manipulate that genome are some of the challenges in the study of parasite persistence and pathogenicity that results in Chagas disease. As the highest impact infectious disease of the Americas with up to 20 million humans and innumerable animals affected, T. cruzi is plagued by poor diagnostic tools, inadequate treatment options and no effective vaccines. New discoveries in all these areas would be greatly facilitated by improved genome editing tools for T. cruzi. We have recently applied CRISPR technology to T. cruzi and demonstrate dramatically increased gene knockout and gene insertion efficiency, providing an unparalleled opportunity for genetic analysis in this important human pathogen. In this project we will further enhance the CRISPR-Cas system in T. cruzi by improving the ability to regulate Cas9 expression, better defining the mechanisms of DNA recombination used to repair Cas9-mediated double-stranded breaks, and using this information to optimize homologous recombination in T. cruzi. Using the demonstrated multiplexing capabilities of CRISPR-Cas9, we will then assess the ability to mutate of large numbers of genes encoding trans-sialidase (ts) molecules in order to evaluate the hypothesis that the ts family of molecules serves an immune evasion function for T. cruzi. This proposal combines the novel use and further development of a powerful genetic system that we demonstrate to be highly efficient in T. cruzi, with an important biological question that up to no was not experimentally tractable. Understanding the role of large gene families in the persistence and pathology of T. cruzi is only possible if we can reduce the number of family members and observe the consequences. Completion of this work is also expected to firmly establish the CRISPR-Cas system as an indispensable tool for the study of T. cruzi.