Bug mapping and fitness testing of chemically synthesized chromosome X

作者：Yi Wu, Bing‐Zhi Li, Meng Zhao, Leslie A. Mitchell, Ze‐Xiong Xie, Qiu-Hui Lin, Xia Wang, Wen-Hai Xiao, Ying Wāng, Xiao Zhou, Hong Liu, Xia Li, Ming-Zhu Ding, Duo Liu, Lu Zhang, Bao-Li Liu, Xiao-Le Wu, Fei-Fei Li, Xiu-Tao Dong, Bin Jia, Wen-Zheng Zhang, Guo-Zhen Jiang, Yue Liu, Xue Bai, Tian-Qing Song, Yan Chen, Si-Jie Zhou, Rui-Ying Zhu, Feng Gao, Zheng Kuang, Xuya Wang, Michael Shen, Kun Yang, Giovanni Stracquadanio, Sarah M. Richardson, Yicong Lin, Lihui Wang, Roy Walker, Yisha Luo, Ping-Sheng Ma, Huanming Yang, Yizhi Cai, Junbiao Dai, Joel S. Bader, Jef D. Boeke, Ying‐Jin Yuan · 发表于：Science · 年份：2017 · DOI：10.1126/science.aaf4706 · 被引用次数：252 · 研究领域：Fungal and yeast genetics research、CRISPR and Genetic Engineering、RNA and protein synthesis mechanisms

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping s...