RNA sequence (RNA-seq) profiling of SynXI strain

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.本研究详述了660千碱基（kilobase, kb）人工合成酵母XI号染色体（synXI）的构建流程，并揭示了非编码DNA（non-coding DNA）元件的人工重设计如何对细胞产生影响。为便于依托合成获得的5~10千碱基DNA片段完成染色体组装，我们开发了基于CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）的体内合成重组交换技术，并借助该技术开展了大规模的缺陷排查、重设计与染色体修复工作，其中包括精准移除200千碱基的意外重复序列。在synXI中，我们将若干生长适配缺陷的根本成因归因于非编码DNA的改造，其中包括与着丝粒（centromere）功能、线粒体（mitochondria）活性相关的缺陷，上述缺陷后续均得到了修复。作为人工合成酵母染色体设计的一部分，我们在大多数基因之间插入了用于Cre重组酶（Cre recombinase）介导重组的loxPsym序列。本研究借助XI号染色体上的GAP1基因座，证实了可通过靶向插入上述序列，按需生成染色体外环状DNA（extrachromosomal circular DNA, eccDNA），从而能够直接研究这类重要分子的功能与复制传播特性。synXI的构建与表征分析揭示了非编码DNA与染色体外环状DNA的相关效应，有助于深化对这类元件的认知，并为后续合成基因组的设计提供参考依据。