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Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

Blount, Benjamin A.; Lu, Xinyu; Driessen, Maureen R. M.; Jovicevic, Dejana; Sanchez, Mateo I; Ciurkot, Klaudia; Zhao, Yu; Lauer, Stephanie; McKiernan, Robert M.; Gowers, Glen-Oliver F.; Sweeney, Fiachra; Fanfani, Viola; Lobzaev, Evgenii; Palacios-Flores, Kim; Walker, Roy; Hesketh, Andy; Oliver, Stephen G.; Cai, Yizhi; Stracquadanio, Giovanni; Mitchell, Leslie A.; Bader, Joel S.; Boeke, Jef D.; Ellis, Tom


Xinyu Lu

Maureen R. M. Driessen

Dejana Jovicevic

Mateo I Sanchez

Klaudia Ciurkot

Yu Zhao

Stephanie Lauer

Robert M. McKiernan

Glen-Oliver F. Gowers

Fiachra Sweeney

Viola Fanfani

Evgenii Lobzaev

Kim Palacios-Flores

Roy Walker

Andy Hesketh

Stephen G. Oliver

Yizhi Cai

Giovanni Stracquadanio

Leslie A. Mitchell

Joel S. Bader

Jef D. Boeke

Tom Ellis


We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.


Blount, B. A., Lu, X., Driessen, M. R. M., Jovicevic, D., Sanchez, M. I., Ciurkot, K., …Ellis, T. Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

Deposit Date Aug 1, 2022
Publicly Available Date Aug 1, 2022
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