Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Abstract

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ~190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes

“…S1). We designed the loxTags for all synthetic chromosomes, including the tRNA neochromosome (Supporting Data S1) which carries rox sites recognized by the Dre recombinase, an orthogonal but not cross acting recombinase system (Anastassiadis et al ., 2009; Schindler et al ., 2022). In this way we can prove the versatility of our tool which can be used with any custom DNA sequence motif.…”

“…However, our results show that SCRaMbLE can have a variety of impacts on yeast karyotype, ranging from segmental to whole chromosome and genome duplications. It was previously reported that aneuploidies and whole genome duplication may have an impact on yeast cells in the presence of hydroxyurea (Schindler et al ., 2022; Shen et al ., 2022). Therefore, we performed growth assays in comparison to the parental strain on solid synthetic complete medium (SC) for the linear and circular strains (Fig.…”

“…In this study, long-read Nanopore sequencing has once more proven its ability to solve highly complex rearranged genomes and seems to be the gold standard to decode the complex genotypes of SCRaMbLE isolates. We have already sequenced up to 24 yeast strains on one MinION flow cell (Luo et al ., 2021; Ong et al ., 2021; Schindler et al ., 2022). Our study confirms previous studies (Shen et al ., 2022) which have shown that circular synthetic chromosomes show a much higher number of Cre-mediated recombination events than linear ones.…”

“…Plug preparation and PFGE was performed as described previously (Schindler et al ., 2022) according to described methods by Hage and Houseley (2013) (Hage and Houseley, 2013) with the following slight alterations. PFGE was undertaken by running samples on a 1.0% agarose gel in 0.5 X TBE solution at 14 °C on a Bio-Rad clamped homogeneous electric field apparatus (CHEF-DR III, BioRad).…”

Visioning synthetic futures for yeast research within the context of current global techno‐political trends

“…The radical re‐engineering approaches of the Sc2.0 consortium have enabled the removal and relocation of all 275 tRNA genes onto a dedicated ‘tRNA neochromosome’ (Schindler et al, 2022). This project has led to unique insights into tRNA, cell and chromosomal biology that would not have been possible through ‘traditional’ approaches.…”