Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics

Benjamin A Blount; 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 S K Walker; Andy Hesketh; Jitong Cai; Stephen G Oliver; Yizhi Cai; Giovanni Stracquadanio; Leslie A Mitchell; Joel S Bader; Jef D Boeke; Tom Ellis

doi:10.1016/j.xgen.2023.100418

Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics

Cell Genom. 2023 Nov 9;3(11):100418. doi: 10.1016/j.xgen.2023.100418. eCollection 2023 Nov 8.

Authors

Benjamin A Blount^{1

2

3}, Xinyu Lu^{1

2}, Maureen R M Driessen^{1

2}, Dejana Jovicevic^{1

2}, Mateo I Sanchez^{1

2}, Klaudia Ciurkot^{1

2}, Yu Zhao⁴, Stephanie Lauer⁴, Robert M McKiernan^{1

2

5}, Glen-Oliver F Gowers^{1

2}, Fiachra Sweeney^{1

5}, Viola Fanfani⁶, Evgenii Lobzaev^{6

7}, Kim Palacios-Flores⁸, Roy S K Walker⁹, Andy Hesketh¹⁰, Jitong Cai¹¹, Stephen G Oliver¹⁰, Yizhi Cai^{6

12}, Giovanni Stracquadanio⁶, Leslie A Mitchell⁴, Joel S Bader¹¹, Jef D Boeke^{4

13}, Tom Ellis^{1

2}

Affiliations

¹ Imperial College Centre for Synthetic Biology, Imperial College London, London, UK.
² Department of Bioengineering, Imperial College London, London, UK.
³ School of Life Sciences, University of Nottingham, Nottingham, UK.
⁴ Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, USA.
⁵ Department of Life Sciences, Imperial College London, London, UK.
⁶ School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.
⁷ School of Informatics, The University of Edinburgh, Edinburgh, UK.
⁸ Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Querétaro, México.
⁹ School of Engineering, Institute for Bioengineering, The University of Edinburgh, Edinburgh, UK.
¹⁰ Department of Biochemistry, University of Cambridge, Cambridge, UK.
¹¹ Department of Biomedical Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA.
¹² Manchester Institute of Biotechnology, University of Manchester, Manchester, UK.
¹³ Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY, USA.

Abstract

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

Keywords: Sc2.0; Yeast 2.0; eccDNA; extrachromosomal circular DNA; genome editing; stress adaptation; synthetic biology; synthetic genomics.