RNA sequence (RNA-seq) profiling of Syn6.5 consolidation strain

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or “CRISPR D-BUGS”, to map phenotypic variants caused by specific designer modifications, known as “bugs”. We first fine-mapped a bug in synthetic chromosome II (synII), and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.Sc2.0项目（Sc2.0 project）正从零开始构建真核生物合成基因组。目前该项目已达成一项重要里程碑：所有Sc2.0单条人工染色体均已完成组装。本研究通过结合先进的核内复制互交技术与tRNA表达盒（tRNA expression cassettes），实现多条合成染色体的整合，成功构建出携带6.5条合成染色体的酵母菌株。研究人员采用Hi-C技术（Hi-C）与长读长直接RNA测序技术，对该菌株的染色体三维组织结构及转录本异构体谱进行了评估。我们开发了CRISPR定向双等位基因URA3辅助基因组扫描技术（CRISPR Directed Biallelic URA3-assisted Genome Scan，简称"CRISPR D-BUGS"），用于定位由特定设计性修饰（即所谓的"设计缺陷（bugs）"）所引发的表型变异。本团队首先对合成染色体II（synII）中的一处设计缺陷进行了精细定位，随后发现了与合成染色体III（synIII）和合成染色体X（synX）相关的组合互作效应，揭示了一种此前未被发现的遗传互作关系，该关联涉及转录调控、肌醇代谢以及tRNASerCGA的丰度调控。最后，为加快整合进程，我们采用染色体替换技术将最大的合成染色体IV（synIV）整合进菌株，最终使单株菌株中整合的Sc2.0基因组占比超过50%。