Synthetic genetic systems for rapid biomolecular evolution in vivo

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。这些系统还具有 开始允许我们使用快速突变积累作为一种在发育过程中追踪细胞谱系的方法 动物。我们的主要成就之一是发明了一种正交DNA复制系统 (正射)。在OrthoRep中，容易出错的正交DNA聚合酶(DNAP)独家复制特殊的 胞质质粒只编码感兴趣的基因(GOIS)，在体内完全驱动它们的持续进化。这 Mira将整合我们实验室在OrthoRep上的工作，并在下一步支持其进一步的开发和应用 五年了。特别是，我们将发展核心OrthoRep技术，以加速GOI在 酵母甚至比我们目前拥有的更多，试图在哺乳动物细胞中建立OrthoRep，以便扩展 OrthoRep可以直接解决的问题范围，将OrthoRep应用于定制机箱的工程 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 希望为这次Mira提出的一系列活动将巩固OrthoRep作为一个异常强大的基因 一种进化酶和蛋白质的系统，能够解决化学、生物、 和生物医学科学。

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. This MIRA 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.