• Reference Type: Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.
  Schindler D, et al. (2023) Design, construction, and functional characterization of a tRNA neochromosome in yeast. Cell 186(24):5237-5253.e22 PMID: 37944512
  • SGD Paper
  • DOI full text
  • PubMed

  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 incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, 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 opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

• Reference Type: Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.
  Zhao Y, et al. (2023) Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions. Cell 186(24):5220-5236.e16 PMID: 37944511
  • SGD Paper
  • DOI full text
  • PubMed

  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 the 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 tRNA_Ser^CGA 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.

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