Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

著者: Yu Zhao, Camila Coelho, Amanda L. Hughes, Luciana Lazar‐Stefanita, Sandy Yang, Aaron N. Brooks, Roy 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. Bader, Giovanni Stracquadanio, Lars M. Steinmetz, Yizhi Cai, Jef D. Boeke - Cell 2023 被引用: 107

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.