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. We perform RNA-sequencing to assess the gene expression changes in the synVIII strain ySLL185 against wild-type strain BY4741.

构建携带数千处定制化修饰的合成酵母染色体，能够以前所未有的尺度探究基因组的灵活性。本研究报道了合成VIII号染色体（synVIII）的组装与表征工作，该染色体可作为对核心功能元件——着丝粒（centromere）开展后续基因组操作的平台。已有研究表明，着丝粒具有独特的特性，因此将着丝粒在单条染色体（VIII号）上的不同位置间进行重定位，可让我们直接测试多个异位位点是否适用于维持着丝粒的功能与稳定性。本研究采用一种基于CRISPR/Cas9的新型方法——单步染色体内着丝粒移植术（Single-Step Intrachromosomal Centromere Transplantation，简称SSICT），对CEN8的118 bp点状着丝粒序列进行移植。无论在野生型还是synVIII菌株中，CEN8的成功移植均会导致VIII号染色体出现非整倍性；而整倍体结构变异提示，若纳入CEN8的左侧旁侧着丝粒序列，则可使异位着丝粒发挥功能。质粒丢失率检测结果显示，特定的左侧旁侧序列可提升携带CEN8的环状微型染色体的稳定性，并降低SSICT过程中非整倍体的发生率。综合以上结果可知，酿酒酵母的最小点状着丝粒在异位移植时不足以维持染色体稳定性，而左侧旁侧的着丝粒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研究提供了重要参考。本研究开展了RNA测序（RNA-sequencing），以评估synVIII菌株ySLL185相较于野生型菌株BY4741的基因表达变化。