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

一、项目摘要/摘要 在过去的五年里，我的实验室在开发能够 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 美国和其他国家快速和可扩展地进化酶、蛋白质和抗体以解决一系列问题 从研究抗药性到按需开发亲和剂。这些系统也已经开始 使我们能够使用快速突变积累作为一种方法来追踪发育中动物的细胞谱系。一 我们的主要成就之一是发明了一种正交DNA复制系统(OrthoRep)。在……里面 OrthoRep是一种易出错的正交DNA聚合酶(DNAP)，它独占复制一种特殊的胞质质粒 只编码感兴趣的基因(GOI)，完全在体内驱动它们的持续进化。母公司米拉资助 我将整合我们实验室在OrthoRep上的工作，并在未来五年支持其进一步开发和应用 好几年了。特别是，我们将发展核心的OrthoRep技术，以加速酵母中GOI的进化 甚至比我们目前更多的是，试图在哺乳动物细胞中建立OrthoRep，以便延长 OrthoRep可以直接解决的一系列问题，将OrthoRep应用到定制机箱的工程中 扩展目标范围和有效性，应用OrthoRep来改进谱系跟踪工具的功能 由我的实验室开发，并将OrthoRep应用于生成相互正交的氨基酰基集合- TRNA合成酶(AARS)/tRNA对支持遗传密码的扩展工作也在我的实验室进行。我们希望 为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 进化中的酶和蛋白质能够解决化学、生物和生物领域的高回报问题 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.