@article{e0d11f8feb2342608a2bbfa82448ca70,
title = "Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions",
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 {\textquoteleft}{\textquoteleft}CRISPR D-BUGS,{\textquoteright}{\textquoteright} to map phenotypic variants caused by specific designer modifications, known as {\textquoteleft}{\textquoteleft}bugs.{\textquoteright}{\textquoteright} 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 tRNASer 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 ",
author = "Yu Zhao and Camila Coelho and Hughes, {Amanda L.} and Luciana Lazar-Stefanita and Sandy Yang and Brooks, {Aaron N.} and Walker, {Roy S. K.} and Weimin Zhang and Stephanie Lauer and Cindy Hernandez and Jitong Cai and Mitchell, {Leslie A.} and Neta Agmon and Yue Shen and Joseph Sall and Viola Fanfani and Anavi Jalan and Jordan Rivera and Feng-Xia Liang and Bader, {Joel S.} and Giovanni Stracquadanio and Steinmetz, {Lars M.} and Yizhi Cai and Boeke, {Jef D.}",
note = "Funding Information: We thank Meru J. Sadhu, Pan Cheng, Guangbin Shi, and Michael Pacold for helpful suggestions. We thank Megan Hogan, Raven Luther, Hannah Ashe, and Gwen Ellis from the Matthew Maurano lab and Runyu Hong from the David Feny{\"o} lab for assistance with WGS analyses. We give special thanks to the Sc2.0 consortium for many discussions and collaborations. This work is part of the Sc2.0 project (http://syntheticyeast.org/), supported by NSF grants MCB-1026068, MCB-1443299, MCB-1616111, and MCB-1921641 to J.D.B. The NYU Microscopy Laboratory is partially supported by the Laura and Isaac Perlmutter Cancer Center support grant NIH/NCI P30CA016087, and the Zeiss Gemini300SEM was purchased with NIH S10 OD019974. Transcriptome profiling was funded by a Volkswagen Stiftung grant (94769) to L.M.S. Figure 5I and the graphical abstract were created with BioRender.com. Conceptualization, Y.Z. L.A.M. and J.D.B.; experiments and data analysis, Y.Z. C.C. A.L.H. L.L.-S. S.Y. A.N.B. R.S.K.W. W.Z. C.H. Y.S. J.S. and V.F.; genomic data organization, J.C. and J.S.B.; manuscript writing, Y.Z. A.L.H. S.Y. S.L. and J.D.B.; supervision, L.A.M. N.A. F.-X.L. G.S. L.M.S. Y.C. and J.D.B.; C.C. S.Y. C.H. A.J. and J.R. are students from the NYU “Build-A-Genome” class. 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. Funding Information: We thank Meru J. Sadhu, Pan Cheng, Guangbin Shi, and Michael Pacold for helpful suggestions. We thank Megan Hogan, Raven Luther, Hannah Ashe, and Gwen Ellis from the Matthew Maurano lab and Runyu Hong from the David Feny{\"o} lab for assistance with WGS analyses. We give special thanks to the Sc2.0 consortium for many discussions and collaborations. This work is part of the Sc2.0 project ( http://syntheticyeast.org/ ), supported by NSF grants MCB-1026068 , MCB-1443299 , MCB-1616111 , and MCB-1921641 to J.D.B. The NYU Microscopy Laboratory is partially supported by the Laura and Isaac Perlmutter Cancer Center support grant NIH/NCI P30CA016087 , and the Zeiss Gemini300SEM was purchased with NIH S10 OD019974 . Transcriptome profiling was funded by a Volkswagen Stiftung grant ( 94769 ) to L.M.S. Figure 5 I and the graphical abstract were created with BioRender.com . Publisher Copyright: {\textcopyright} 2023 The Authors",
year = "2023",
month = nov,
day = "22",
doi = "10.1016/j.cell.2023.09.025",
language = "English",
volume = "186",
pages = "5220--5236.e16",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "24",
}