RNA sequence (RNA-seq) profiling of SynVIII strain

Building synthetic yeast chromosomes with thousands of designer modifications empowers probing of genome flexibility on an unprecedented scale. Here, we describe the assembly and characterization of synthetic chromosome VIII (synVIII), which served as a platform for further genome manipulation of an essential functional element, the centromere. Recent findings suggest that centromeres may have distinct characteristics and thus relocating the centromere to various positions along a single chromosome (VIII) enables us to directly test the suitability of several ectopic destinations for centromere function and stability. We used a novel CRISPR/Cas9-mediated approach, Single-Step Intrachromosomal Centromere Transplantation (SSICT), to relocate the 118 bp point centromere sequence of CEN8. Successful transplantation of CEN8 invariably resulted in chromosome VIII aneuploidy regardless of ectopic centromere position in wild-type or synVIII strains and euploid structural variants hinted that including left-flanking pericentromeric sequences of CEN8 allowed ectopic function. Plasmid loss rate assays revealed that specific left-flanking sequences increased stability of a CEN8-containing circular minichromosome and reduced incidence of aneuploidy during SSICT. Together, these results show that the minimal point centromere of budding yeast is insufficient to confer chromosome stability when transplanted ectopically, and is rescued by left-flanking pericentromeric sequence. This is an important consideration for future genome engineering projects involving transplantation of functional elements.构建携带数千处定制化修饰的合成酵母染色体，可在前所未有的研究尺度下探究基因组的灵活性。本研究报道了合成八号染色体（synthetic chromosome VIII, synVIII）的组装与表征工作，以此作为平台对核心功能元件——着丝粒（centromere）开展后续基因组操作。近期研究表明，着丝粒具有独特的序列特征，因此将着丝粒沿八号染色体的不同位置进行异位重定位，可直接测试多个异位位点支持着丝粒功能与稳定性的适配性。本研究采用一种新型CRISPR/Cas9介导的单步染色体内着丝粒移植（Single-Step Intrachromosomal Centromere Transplantation, SSICT）技术，对酿酒酵母CEN8的118 bp点着丝粒序列（point centromere sequence）进行重定位。无论在野生型还是synVIII菌株中，无论异位着丝粒位置如何，成功移植CEN8均会导致八号染色体发生非整倍体（aneuploidy）；而整倍体结构变异（euploid structural variants）提示，包含CEN8左侧着丝粒旁侧序列可使异位着丝粒发挥功能。质粒丢失率检测（plasmid loss rate assays）结果显示，特定的左侧旁侧序列可提升携带CEN8的环状微型染色体（circular minichromosome）的稳定性，并降低SSICT过程中非整倍体的发生率。综上，本研究结果表明，酿酒酵母的最小点着丝粒序列异位移植后不足以维持染色体稳定性，而左侧着丝粒旁侧序列可挽救这一缺陷。这一发现为未来涉及功能元件移植的基因组工程（genome engineering）项目提供了重要参考依据。