Equipment Supplement for "Synthetic genetic systems for rapid biomolecular evolution in vivo"

I.项目概要/摘要 在过去的五年里，我的实验室在基因系统的开发方面取得了重大进展， 在体内驱动用户选择的感兴趣基因的快速突变和进化。这些系统允许 我们和其他人快速和可扩展地进化酶，蛋白质和抗体，以解决一系列问题 从研究耐药性到按需制造亲和试剂。这些系统也开始 使我们能够使用快速突变积累作为追踪发育中动物细胞谱系的方法。一 我们的主要成就之一是发明了正交DNA复制系统（OrthoRep）。在 OrthoRep是一种容易出错的正交DNA聚合酶（DNAP），专门复制特殊的胞质质粒 仅编码感兴趣的基因（GOI），完全在体内驱动它们的持续进化。父母MIRA补助金 在未来的五年里，我将整合我们实验室在OrthoRep上的工作，并支持其进一步的开发和应用 年特别是，我们将发展核心OrthoRep技术，以加速GOI在酵母中的进化 甚至比我们目前所做的更多，试图在哺乳动物细胞中建立OrthoRep，以延长 OrthoRep可以直接解决的一系列问题，将OrthoRep应用于定制Cas9的工程设计， 扩展定位的范围和有效性，应用OrthoRep来提高谱系追踪工具的能力 我的实验室开发的，并应用OrthoRep生成相互正交的氨酰- tRNA合成酶（阿尔斯）/tRNA对，以支持遗传密码扩展的努力也正在我的实验室。我们希望 为该MIRA提出的一系列活动将巩固OrthoRep作为一个非常强大的遗传系统， 进化酶和蛋白质，能够解决化学、生物和生物学领域的高回报问题， 生物医学科学

I. Project Summary/Abstract Over the past five years, my lab has made significant strides in the development of genetic systems capable of driving the rapid mutation and evolution of user-selected genes of interest in vivo. These systems have allowed us and others to quickly and scalably evolve enzymes, proteins, and antibodies to address a range of problems spanning from studying drug resistance to creating affinity reagents on demand. These systems have also begun to allow us to use rapid mutational accumulation as a method for tracing cell lineage in developing animals. One of our key accomplishments has been the invention of an orthogonal DNA replication system (OrthoRep). In OrthoRep, an error-prone orthogonal DNA polymerase (DNAP) exclusively replicates a special cytosolic plasmid encoding only genes of interest (GOIs), driving their continuous evolution fully in vivo. The parent MIRA grant will integrate our lab’s work on OrthoRep and support its further development and application in the next five years. In particular, we will grow the core OrthoRep technology in order to accelerate GOI evolution in yeast even more than we currently have, attempt to establish OrthoRep in mammalian cells in order to extend the range of problems OrthoRep can directly address, apply OrthoRep to the engineering of bespoke Cas9s to extend the range and efficacy of targeting, apply OrthoRep to improve the capabilities of a lineage tracing tool developed by my lab, and apply OrthoRep to the generation of mutually orthogonal collections of aminoacyl- tRNA synthetase (aaRS)/tRNA pairs to support genetic code expansion efforts also ongoing in my lab. We hope the set of activities proposed for this MIRA will solidify OrthoRep as an exceptionally powerful genetic system for evolving enzymes and proteins capable of solving high-reward problems in the chemical, biological, and biomedical sciences.