Engineering the ribosomal DNA in a megabase synthetic chromosome

• DOI: 10.1126/science.aaf3981
• Publication Date: 2017-07-11T17:30:19Z
• Authors: Weimin Zhang; Guanghou Zhao; Zhouqing Luo; Yicong Lin; Lihui Wang; Yakun Guo; Ann Wang; Shuangying Jiang; Qingwen Jiang; Jianhui Gong; Yun Wang; Sha Hou; Jing Huang; Tianyi Li; Yiran Qin; Junkai Dong; Qin Qin; Jiaying Zhang; Xinzhi Zou; Xi He; Li Zhao; Yibo Xiao; Meng Xu; Erchao Cheng; Ning Huang; Tong Zhou; Yue Shen; Roy Walker; Yisha Luo; Zheng Kuang; Leslie A. Mitchell; Kun Yang; Sarah M. Richardson; Yi Wu; Bing-Zhi Li; Ying-Jin Yuan; Huanming Yang; Jiwei Lin; Guo-Qiang Chen; Qingyu Wu; Joel S. Bader; Yizhi Cai; Jef D. Boeke; Junbiao Dai
• Keywords: Yeast artificial chromosome

INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, goal that may not realized anytime soon. Nonetheless, with advances DNA synthesis technology, complete genetic material of organism now synthesized chemically. Hitherto, genomes several organisms including viruses, phages, and bacteria have designed constructed. These synthetic are able to direct all normal biological functions, capable self-replication production offspring. Several years ago, group scientists worldwide formed international consortium reconstruct genome budding yeast, Saccharomyces cerevisiae . RATIONALE The yeast genome, designated Sc2.0, was according set arbitrary rules, elimination transposable elements incorporation specific facilitate further manipulation. Among 16 S. chromosomes, chromosome XII is unique as one longest chromosomes (~1 million base pairs) additionally encodes highly repetitive ribosomal locus, which forms well-organized nucleolus. We report on design, construction, characterization XII, physically largest cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, based native sequence , chemically synthesized. SynXII assembled using two-step method involving, successive megachunk integration produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding full-length functional Minor growth defect “bugs” detected synXII were caused deletion tRNA genes corrected introducing ectopic copy single gene. gene cluster (rDNA) left intact during assembly process subsequently replaced modified rDNA unit. same unit also used regenerate at three distinct chromosomal locations. signature sequences internal transcribed spacer (ITS), often determine species identity standard barcoding procedures, swapped generate strain would identified bayanus. Remarkably, these substantial changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION locus plastic; only it moved other loci, altered its ITS region masquerade defined barcoding, widely taxonomy. ability perform “species morphing” reported here presumably reflects degree evolutionary flexibility regions change. However, this clearly infinitely flexible, relatively modest intragenus tolerated. More severe intergenus differences did result rDNAs, probably because defects rRNA processing. build, debug megabase-sized together position, speaks remarkable overall genome. Hierarchical subsequent restructuring synXII. two steps: First, strains built segments corresponding designer sequences. Next, combined withmultiple rounds ofmating/sporulation, eventually generating encoding fulllength synXII.The repeats removed, modified, regenerated locations for morphing restructuring.