Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27514, USA.
Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27514, USA.
Cell. 2019 Jul 25;178(3):748-761.e17. doi: 10.1016/j.cell.2019.05.051. Epub 2019 Jul 4.
Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.
定向进化，即朝着预定目标进行人工选择，是开发新的分子工具和治疗方法的常用手段。成功的定向分子进化活动反复测试具有设计选择压力的不同序列。单细胞生物及其病毒病原体在这方面非常出色，已经使用了几十年。然而，许多定向进化的理想目标在单细胞背景下表现不佳或不自然。在这里，我们提出了一种在哺乳动物细胞中进行简便定向进化的系统。我们使用 RNA 噬菌体辛德毕斯作为遗传和多样性的载体，实现了超过 10 个碱基每代 24 小时的选择循环。通过在宿主细胞内的遗传激活序列来实现选择，因此该系统被命名为：遗传激活序列的病毒进化，或“ VEGAS ”。使用 VEGAS ，我们在不到 1 周的时间内针对功能性信号终点进化转录因子、GPCR 和变构纳米抗体。
