Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 37944511.
- Also identified by DOI 10.1016/j.cell.2023.09.025.
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Abstract
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<sub>Ser</sub><sup>CGA</sup> 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.
Medical subject headings
- Chromosomes, Artificial, Yeast
- Saccharomyces cerevisiae
- Genome, Fungal