Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Yu Zhao, Camila Coelho, Amanda L. Hughes, Luciana Lazar-Stefanita, Sandy Yang, Aaron N. Brooks, Roy S.K. Walker, Weimin Zhang, Stephanie Lauer, Cindy Hernandez, Jitong Cai, Leslie A. Mitchell, Neta Agmon, Yue Shen, Joseph Sall, Viola Fanfani, Anavi Jalan, Jordan Rivera, Feng Xia Liang, Joel S. BaderGiovanni Stracquadanio, Lars M. Steinmetz, Yizhi Cai, Jef D. Boeke

Research output: Contribution to journalArticlepeer-review


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 tRNASerCGA 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.

Original languageEnglish (US)
Pages (from-to)5220-5236.e16
Issue number24
StatePublished - Nov 22 2023


  • 3D organization
  • Sc2.0
  • chromosome substitution
  • combinatorial interactions
  • consolidation
  • synthetic chromosomes
  • transcript isoforms

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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