Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

Laura H McCulloch; Vijayan Sambasivam; Amanda L Hughes; Narayana Annaluru; Sivaprakash Ramalingam; Viola Fanfani; Evgenii Lobzaev; Leslie A Mitchell; Jitong Cai; Build-A-Genome Class; Hua Jiang; John LaCava; Martin S Taylor; William R Bishai; Giovanni Stracquadanio; Lars M Steinmetz; Joel S Bader; Weimin Zhang; Jef D Boeke; Srinivasan Chandrasegaran

doi:10.1016/j.xgen.2023.100419

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

Cell Genom. 2023 Nov 9;3(11):100419. doi: 10.1016/j.xgen.2023.100419. eCollection 2023 Nov 8.

Authors

Laura H McCulloch¹, Vijayan Sambasivam², Amanda L Hughes³, Narayana Annaluru², Sivaprakash Ramalingam², Viola Fanfani⁴, Evgenii Lobzaev^{4

5}, Leslie A Mitchell¹, Jitong Cai⁶; Build-A-Genome Class; Hua Jiang⁷, John LaCava^{7

8}, Martin S Taylor⁹, William R Bishai¹⁰, Giovanni Stracquadanio⁴, Lars M Steinmetz^{3

11

12}, Joel S Bader^{6

13}, Weimin Zhang¹, Jef D Boeke^{1

13

14}, Srinivasan Chandrasegaran²

Affiliations

¹ Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.
² Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
³ European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany.
⁴ School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK.
⁵ School of Informatics, The University of Edinburgh, Edinburgh EH8 9AB, UK.
⁶ Department of Biomedical Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
⁷ Laboratory of Cellular and Structural Biology, Rockefeller University, New York, NY 10065, USA.
⁸ European Research Institute for the Biology of Ageing, University Medical Center Groningen, Groningen, the Netherlands.
⁹ Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
¹⁰ Department of Medicine/Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
¹¹ Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
¹² Department of Genetics, School of Medicine, Stanford University, Stanford, CA 94305, USA.
¹³ High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
¹⁴ Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, USA.

Abstract

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Keywords: EST3; Saccharomyces cerevisiae; centromere destabilization; chromoduction; chromosome debugging; chromosome substitution; megachunk assembly; synIX; transcriptomics.