Molecular mechanisms and applications of monomeric DNA endonucleases

单体DNA核酸内切酶的分子机制及应用

• 批准号: RGPIN-2015-04800
• 负责人: Edgell, David
• 金额: $ 3.79万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=649924
• 关键词: Molecular; mechanisms; applications; monomeric; DNA

My program of research deals with mobile genetic elements, discrete units of DNA that promote their own spread within and between genomes. For the past ten years, my laboratory has studied the evolution and function of homing endonucleases, site-specific DNA endonucleases that spread by making a double-strand break (or nick) in a DNA target. Over the last funding period, we focused on basic mechanistic questions concerning DNA cleavage by the GIY-YIG family of homing endonucleases, and translating this mechanistic understanding to the generation of novel monomeric chimeric nucleases for genome-editing applications. Academic and industrial researchers use genome-editing reagents to introduce precise modifications in complex genomes for applications in agriculture, aquaculture, synthetic biology, and discovery-based research in model organisms. ******Our long-term goal is to develop a set of monomeric nucleases with tailored cleavage specificities for genome editing and synthetic biology applications that require high fidelity. Monomeric domains are useful for genome-engineering applications because a monomeric nuclease domain requires that only a single chimeric nuclease need be designed to target a given sequence. Moreover, many monomeric nuclease domains have inherent cleavage specificity that will limit off-target cleavage and cellular toxicity. Understanding the molecular basis of cleavage specificity will enable more predictable targeting of the domains to desired sequences, as well as identifying potential off-target sites. This will be achieved by a combination of genetic selections and protein-DNA crosslinking studies to identify residues that contribute to DNA cleavage specificity. For the GIY-YIG enzymes, a long term goal will be structural studies of GIY-YIG homing endonucleases bound to a DNA substrate, as this will give us insight into residues that contribute to cleavage specificity. Towards this goal, we have initiated a collaboration with Dr. Murray Junop (Western University). ******In summary, my research program addresses fundamental questions concerning structure and function of site-specific DNA endonucleases, in addition to biotechnological applications that derive from these studies. Given the widespread use of genome-editing nucleases in academic and industrial settings, our research program is well positioned to make important contributions in the development of critical reagents. *****

我的研究计划涉及移动遗传元素，即DNA的离散单位，这些单位在基因组之间和基因组之间促进自身传播。在过去的十年中，我的实验室研究了归核核酸内切酶的演变和功能，特定于位点的DNA核酸内切酶通过在DNA靶中进行双链断裂（或刻度）来扩散。在上一个融资期间，我们重点介绍了有关归巢核酸内切核酸内核酸内切酶DNA裂解的基本机制问题，并将这种机械理解转化为用于基因组编辑应用的新型单体嵌合核酸酶的产生。学术和工业研究人员使用基因组编辑的试剂在复杂的基因组中引入用于农业，水产养殖，合成生物学和基于发现的模型生物体研究的精确修饰。 *****我们的长期目标是开发一组单核核酸酶，具有针对需要高保真度的基因组编辑和合成生物学应用的定制切割特异性。单体结构域对基因组工程应用有用，因为单体核酸酶结构域要求仅设计单个嵌合核酸酶来靶向给定序列。此外，许多单体核酸酶结构域具有固有的切割特异性，该特异性将限制靶向裂解和细胞毒性。了解切割特异性的分子基础将使域更可预测的靶向所需的序列，并识别潜在的脱离靶向位点。这将通过遗传选择和蛋白-DNA交联研究的结合来实现，以鉴定有助于DNA裂解特异性的残基。对于GIY-YIG酶，长期目标将是与DNA底物结合的GIY-YIG HOHING核酸内切酶的结构研究，因为这将使我们深入了解有助于切割特异性的残基。为了实现这一目标，我们已经与默里·朱诺普（Murray Junop）博士（西部大学）合作。 ******总而言之，我的研究计划还解决了有关位点特异性DNA核酸内切酶的结构和功能的基本问题，此外，除了从这些研究中得出的生物技术应用外。鉴于在学术和工业环境中广泛使用基因组编辑的核酸酶，我们的研究计划在关键试剂的开发中做出了重要的贡献。 *****