Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

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: jef.boeke@nyulangone.org.

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

Free article

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Yu Zhao et al. Cell. 2023.

Free article

. 2023 Nov 22;186(24):5220-5236.e16.

doi: 10.1016/j.cell.2023.09.025. Epub 2023 Nov 8.

Authors

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: jef.boeke@nyulangone.org.

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.

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Conflict of interest statement

Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., and a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Logomix Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute.

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Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Affiliations

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Authors

Affiliations

Abstract

Conflict of interest statement

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials