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 Bader; Giovanni Stracquadanio; Lars M Steinmetz; Yizhi Cai; Jef D Boeke

doi:10.1016/j.cell.2023.09.025

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Cell. 2023 Nov 22;186(24):5220-5236.e16. doi: 10.1016/j.cell.2023.09.025. Epub 2023 Nov 8.

Authors

Yu Zhao¹, Camila Coelho¹, Amanda L Hughes², Luciana Lazar-Stefanita¹, Sandy Yang¹, Aaron N Brooks², Roy S K Walker³, Weimin Zhang¹, Stephanie Lauer¹, Cindy Hernandez¹, Jitong Cai⁴, Leslie A Mitchell¹, Neta Agmon¹, Yue Shen⁵, Joseph Sall⁶, Viola Fanfani⁷, Anavi Jalan⁸, Jordan Rivera⁸, Feng-Xia Liang⁶, Joel S Bader⁴, Giovanni Stracquadanio⁷, Lars M Steinmetz⁹, Yizhi Cai¹⁰, Jef D Boeke¹¹

Affiliations

¹ Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.
² European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany.
³ School of Engineering, Institute for Bioengineering, the University of Edinburgh, Edinburgh EH9 3BF.
⁴ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
⁵ BGI, Shenzhen, Beishan, Industrial Zone, Shenzhen 518083, China; Guangdong Provincial Key Laboratory of Genome Read and Write, BGI, Shenzhen, Shenzhen 518120, China.
⁶ Microscopy Laboratory, NYU Langone Health, New York, NY 10016, USA.
⁷ School of Biological Sciences, the University of Edinburgh, Edinburgh EH9 3BF.
⁸ Department of Biology, New York University, New York, NY, USA.
⁹ European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany; Department of Genetics and Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
¹⁰ Manchester Institute of Biotechnology, the University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
¹¹ Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA; Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, New York, NY 11201, USA. Electronic address: [email protected].

PMID: 37944511
DOI: 10.1016/j.cell.2023.09.025

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_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.

Keywords: 3D organization; CRISPR D-BUGS; Sc2.0; chromosome substitution; combinatorial interactions; consolidation; synthetic chromosomes; transcript isoforms.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Base Sequence
Chromosomes / genetics
Chromosomes, Artificial, Yeast*
Genome, Fungal*
Saccharomyces cerevisiae* / genetics
Synthetic Biology

Abstract

Publication types

MeSH terms

Grants and funding