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. *****

我的研究项目涉及移动遗传元件，即促进其在基因组内部和基因组之间传播的离散 DNA 单元。在过去的十年里，我的实验室研究了归巢核酸内切酶的进化和功能，这些酶是通过在 DNA 靶标中产生双链断裂（或切口）来传播的位点特异性 DNA 核酸内切酶。在上一个资助期间，我们专注于有关归巢核酸内切酶 GIY-YIG 家族切割 DNA 的基本机制问题，并将这种机制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生成。学术和工业研究人员使用基因组编辑试剂对复杂基因组进行精确修饰，以应用于农业、水产养殖、合成生物学和模式生物的基于发现的研究。 ******我们的长期目标是开发一套具有定制切割特异性的单体核酸酶，用于需要高保真度的基因组编辑和合成生物学应用。单体结构域可用于基因组工程应用，因为单体核酸酶结构域要求仅需要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，这将限制脱靶切割和细胞毒性。了解切割特异性的分子基础将使域能够更可预测地靶向所需序列，并识别潜在的脱靶位点。这将通过结合遗传选择和蛋白质-DNA 交联研究来识别有助于 DNA 切割特异性的残基来实现。对于 GIY-YIG 酶，长期目标是对与 DNA 底物结合的 GIY-YIG 归巢核酸内切酶进行结构研究，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，我们发起了与 Murray Junop 博士（西方大学）的合作。 ******总之，我的研究计划除了从这些研究中衍生出的生物技术应用之外，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。鉴于基因组编辑核酸酶在学术和工业环境中的广泛使用，我们的研究项目完全有能力为关键试剂的开发做出重要贡献。 *****