Hi-C 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 Hi-C to assess whether two centromere copies in the SynVIII strain was structurally functional (Hi-C).