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

 描述(申请人提供)：锥虫克氏锥虫基因组的复杂性和独特性，以及操纵该基因组的工具相对缺乏，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，克氏锥虫诊断工具不佳，治疗选择不足，也没有有效的疫苗。克氏锥虫基因组编辑工具的改进将极大地促进所有这些领域的新发现。我们最近已经将CRISPR技术应用于克氏锥虫，并展示了显著提高的基因敲除和基因插入效率，为这种重要的人类病原体的基因分析提供了一个无与伦比的机会。在本项目中，我们将通过提高调节Cas9表达的能力，更好地确定修复Cas9介导的双链断裂的DNA重组机制，并利用这些信息来优化T.ruzi中的同源重组，进一步增强T.ruzi中的CRISPR-Cas系统。利用CRISPR-Cas9的多路复用能力，我们将评估编码反式唾液酸酶(Ts)分子的大量基因突变的能力，以评估ts家族分子对克氏锥虫具有免疫逃避功能的假设。这一建议结合了强大的遗传系统的新用途和进一步发展，我们证明该系统在克氏锥虫中是高效的，以及一个重要的生物学问题，在实验上不容易处理。只有减少家族成员的数量并观察其后果，才有可能理解大基因家族在克氏锥虫的持久性和病理学中的作用。这项工作的完成还有望使CRISPR-CAS系统牢固地成为研究克氏锥虫不可或缺的工具。

 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.