Molecular mechanisms and applications of monomeric DNA endonucleases

我的研究项目涉及可移动的遗传元素，即促进自身在基因组内和基因组之间传播的离散DNA单元。在过去的十年里，我的实验室一直在研究归宿内切酶的进化和功能，这是一种通过在DNA靶标上造成双链断裂(或缺口)来传播的定点DNA内切酶。在过去的资助期间，我们专注于关于GIY-YIG家族定位内切酶切割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在复杂基因组中引入精确的修饰，用于农业、水产养殖、合成生物学和模式生物中基于发现的研究。*我们的长期目标是开发一套具有量身定制的切割特异性的单体核酸酶，用于基因组编辑和要求高保真的合成生物学应用。单体结构域对于基因组工程应用是有用的，因为单体核酸酶结构域要求只需要设计一个嵌合核酸酶来针对给定的序列。此外，许多单体核酸酶结构域具有固有的切割专一性，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结构域能够更可预测地靶向所需的序列，以及识别潜在的脱靶位点。这将通过基因选择和蛋白质-DNA交联研究相结合的方式来实现，以确定有助于DNA切割特异性的残基。对于GIY-YIG酶，长期目标将是研究与DNA底物结合的GIY-YIG归巢内切酶的结构，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。总而言之，除了这些研究衍生的生物技术应用外，我的研究计划还解决了与特定位点DNA内切酶的结构和功能有关的基本问题。鉴于基因组编辑核酸酶在学术和工业环境中的广泛使用，我们的研究计划处于有利地位，可以在关键试剂的开发中做出重要贡献。*****

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. *****