TY - JOUR
T1 - Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics
AU - Blount, Benjamin A.
AU - Lu, Xinyu
AU - Driessen, Maureen R. M.
AU - Jovicevic, Dejana
AU - Sanchez, Mateo I.
AU - Ciurkot, Klaudia
AU - Zhao, Yu
AU - Lauer, Stephanie
AU - McKiernan, Robert M.
AU - Gowers, Glen-Oliver F.
AU - Sweeney, Fiachra
AU - Fanfani, Viola
AU - Lobzaev, Evgenii
AU - Palacios-Flores, Kim
AU - Walker, Roy S. K.
AU - Hesketh, Andy
AU - Cai, Jitong
AU - Oliver, Stephen G.
AU - Cai, Yizhi
AU - Stracquadanio, Giovanni
AU - Mitchell, Leslie A.
AU - Bader, Joel S.
AU - Boeke, Jef D.
AU - Ellis, Tom
N1 - Funding Information:
We would like to thank Paul Freemont, Alistair Elfick, Ian Roberts, and Anil Wipat for their support and advice; William Shaw for his help in establishing CRISPR gap repair methods; and Markus Ralser for his gift of plasmid pHLUM. This work was funded by BBSRC awards BB/K019791/1 and BB/R002614/1. B.A.B. was supported by the University of Nottingham through a Nottingham Research Fellowship. Conceptualization, B.A.B. A.H. S.G.O. L.A.M. J.S.B. J.D.B. and T.E.; methodology, B.A.B. Y.Z. S.L. G.S. L.A.M. J.D.B. and T.E.; software, R.M.M. G.-O.F.G. S.G.O. L.A.M. and J.S.B.; validation, B.A.B. M.I.S. Y.Z. S.L. G.-O.F.G. and K.P.-F.; formal analysis, B.A.B. X.L. K.C. V.F. E.L. K.P.-F. and T.E.; investigation, B.A.B. X.L. M.R.M.D. D.J. M.I.S. K.C. Y.Z. S.L. and F.S.; resources, B.A.B. R.S.K.W. Y.C. G.S. L.A.M. J.S.B. J.D.B. and T.E.; data curation, B.A.B. K.C. R.M.M. G.-O.F.G. V.F. E.L. J.C. G.S. and T.E.; writing – original draft, B.A.B.; writing – review & editing, B.A.B. Y.Z. S.L. S.G.O. G.S. J.S.B. J.D.B. and T.E.; visualization, B.A.B. X.L. and M.I.S.; supervision, B.A.B. S.G.O. Y.C. G.S. J.S.B. J.D.B. and T.E.; project administration, B.A.B. J.D.B. and T.E.; funding acquisition, B.A.B. S.G.O. J.D.B. and T.E. B.A.B. is a scientific advisory board (SAB) member of Eden Bio Ltd. T.E. is a consultant to Replay Holdings, LLC and SAB member of Modern Synthesis, Inc. J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; a consultant to Opentrons Labworks, Inc.; a founder and SAB member of and consultant to ReOpen Diagnostics, LLC; and serves or served on the SAB of the following: Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. L.A.M. is a founder of Neochromosome, Inc. and an employee of Opentrons Labworks, Inc.
Funding Information:
We would like to thank Paul Freemont, Alistair Elfick, Ian Roberts, and Anil Wipat for their support and advice; William Shaw for his help in establishing CRISPR gap repair methods; and Markus Ralser for his gift of plasmid pHLUM. This work was funded by BBSRC awards BB/K019791/1 and BB/R002614/1 . B.A.B. was supported by the University of Nottingham through a Nottingham Research Fellowship .
Publisher Copyright:
© 2023 The Author(s)
PY - 2023/11/8
Y1 - 2023/11/8
N2 - We describe construction of the synthetic yeast chromosomeXI (synXI) and reveal the effects of redesign at non-coding DNA elements. The660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled fromsynthesized DNA fragments before CRISPR-based methods were used in a process ofbug discovery, redesign, and chromosome repair, including precise compaction of200 kb of repeat sequence. Repaired defects were related to poor centromerefunction and mitochondrial health and were associated with modifications tonon-coding regions. As part of the Sc2.0 design, loxPsym sequences forCre-mediated recombination are inserted between most genes. Using the GAP1locus from chromosome XI, we show that these sites can facilitate inducedextrachromosomal circular DNA (eccDNA) formation, allowing direct study of theeffects and propagation of these important molecules. Construction andcharacterization of synXI contributes to our understanding of non-coding DNAelements, provides a useful tool for eccDNA study, and will inform futuresynthetic genome design.
AB - We describe construction of the synthetic yeast chromosomeXI (synXI) and reveal the effects of redesign at non-coding DNA elements. The660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled fromsynthesized DNA fragments before CRISPR-based methods were used in a process ofbug discovery, redesign, and chromosome repair, including precise compaction of200 kb of repeat sequence. Repaired defects were related to poor centromerefunction and mitochondrial health and were associated with modifications tonon-coding regions. As part of the Sc2.0 design, loxPsym sequences forCre-mediated recombination are inserted between most genes. Using the GAP1locus from chromosome XI, we show that these sites can facilitate inducedextrachromosomal circular DNA (eccDNA) formation, allowing direct study of theeffects and propagation of these important molecules. Construction andcharacterization of synXI contributes to our understanding of non-coding DNAelements, provides a useful tool for eccDNA study, and will inform futuresynthetic genome design.
U2 - 10.1016/j.xgen.2023.100418
DO - 10.1016/j.xgen.2023.100418
M3 - Article
C2 - 38020971
SN - 2666-979X
VL - 3
JO - Cell Genomics
JF - Cell Genomics
IS - 11
M1 - 100418
ER -