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

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

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.

Original language	English
Article number	100419
Number of pages	24
Journal	Cell Genomics
Volume	3
Issue number	11
DOIs	https://doi.org/10.1016/j.xgen.2023.100419
Publication status	Published - 8 Nov 2023

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10.1016/j.xgen.2023.100419

1-s2.0-S2666979X23002458-mainFinal published version, 4.96 MBLicence: CC BY-NC-ND

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McCulloch, L. H., Sambasivam, V., Hughes, A. L., Annaluru, N., Ramalingam, S., Fanfani, V., Lobzaev, E., Mitchell, L. A., Cai, J., Build-A-Genome Class, Jiang, H., LaCava, J., Taylor, M. S., Bishai, W. R., Stracquadanio, G., Steinmetz, L. M., Bader, J. S., Zhang, W., Boeke, J. D., & Chandrasegaran, S. (2023). Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains. Cell Genomics, 3(11), Article 100419. https://doi.org/10.1016/j.xgen.2023.100419

@article{d216bda09ec7478c89007f881f26e97f,

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

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. ",

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

note = "Funding Information: We thank Meghan O{\textquoteright}Keefe for collecting the BAG student authors{\textquoteright} information. The work reported here forms a portion of L.H.M.{\textquoteright}s PhD thesis. We thank the NSF for grants MCB-1026068 , MCB-1443299 , MCB-1616111 , and MCB-1921641 to J.D.B. and MCB-1445545 to J.S.B., which support this work. Funding Information: We thank Meghan O'Keefe for collecting the BAG student authors{\textquoteright} information. The work reported here forms a portion of L.H.M.{\textquoteright}s PhD thesis. We thank the NSF for grants MCB-1026068, MCB-1443299, MCB-1616111, and MCB-1921641 to J.D.B. and MCB-1445545 to J.S.B. which support this work. J.D.B. S.C. and W.Z. conceptualized the study. W.Z. S.C. L.H.M. and J.D.B. designed the experiments. J.D.B. and J.S.B. designed synIX. The Build-A-Genome class members constructed building blocks. V.S. N.A. and S.R. constructed minichunks and incorporated them for megachunks A–F. L.H.M. designed, constructed, and delivered megachunks G–I and performed error correction, bug mapping, bug correction, and functional studies of synIX. L.H.M. and W.Z. developed and evaluated the chromosome substitution method. L.H.M. A.L.H. J.C. V.F. E.L. and G.S. performed sequencing and data analyses. L.H.M. W.Z. and J.D.B. wrote the manuscript. L.A.M. H.J. J.L. M.S.T. and W.R.B. provided advice. S.C. J.D.B. J.S.B. and L.M.S. provided lab supervision. All authors reviewed and edited the manuscript. J.D.B. is a founder and director of CDI Labs, Inc. a founder of and consultant to Neochromosome, Inc, a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the scientific advisory board of the following: Logomix, Inc. Sangamo, Inc. Modern Meadow, Inc. Rome Therapeutics, Inc. Sample6, Inc. Tessera Therapeutics, Inc. and the Wyss Institute. J.S.B. is a founder of Neochromosome, Inc. consultant to Opentrons Labworks, Inc. and advisor to Reflexion Pharmaceuticals, Inc. Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = nov,

day = "8",

doi = "10.1016/j.xgen.2023.100419",

language = "English",

volume = "3",

journal = "Cell Genomics",

issn = "2666-979X",

publisher = "Cell Press",

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McCulloch, LH, Sambasivam, V, Hughes, AL, Annaluru, N, Ramalingam, S, Fanfani, V, Lobzaev, E, Mitchell, LA, Cai, J, Build-A-Genome Class, Jiang, H, LaCava, J, Taylor, MS, Bishai, WR, Stracquadanio, G, Steinmetz, LM, Bader, JS, Zhang, W, Boeke, JD & Chandrasegaran, S 2023, 'Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains', Cell Genomics, vol. 3, no. 11, 100419. https://doi.org/10.1016/j.xgen.2023.100419

TY - JOUR

T1 - Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

AU - McCulloch, Laura H.

AU - Sambasivam, Vijayan

AU - Hughes, Amanda L.

AU - Annaluru, Narayana

AU - Ramalingam, Sivaprakash

AU - Fanfani, Viola

AU - Lobzaev, Evgenii

AU - Mitchell, Leslie A

AU - Cai, Jitong

AU - Build-A-Genome Class

AU - Jiang, Hua

AU - LaCava, John

AU - Taylor, Martin S.

AU - Bishai, William R.

AU - Stracquadanio, Giovanni

AU - Steinmetz, Lars M.

AU - Bader, Joel S.

AU - Zhang, Weimin

AU - Boeke, Jef D.

AU - Chandrasegaran, Srinivasan

N1 - Funding Information: We thank Meghan O’Keefe for collecting the BAG student authors’ information. The work reported here forms a portion of L.H.M.’s PhD thesis. We thank the NSF for grants MCB-1026068 , MCB-1443299 , MCB-1616111 , and MCB-1921641 to J.D.B. and MCB-1445545 to J.S.B., which support this work. Funding Information: We thank Meghan O'Keefe for collecting the BAG student authors’ information. The work reported here forms a portion of L.H.M.’s PhD thesis. We thank the NSF for grants MCB-1026068, MCB-1443299, MCB-1616111, and MCB-1921641 to J.D.B. and MCB-1445545 to J.S.B. which support this work. J.D.B. S.C. and W.Z. conceptualized the study. W.Z. S.C. L.H.M. and J.D.B. designed the experiments. J.D.B. and J.S.B. designed synIX. The Build-A-Genome class members constructed building blocks. V.S. N.A. and S.R. constructed minichunks and incorporated them for megachunks A–F. L.H.M. designed, constructed, and delivered megachunks G–I and performed error correction, bug mapping, bug correction, and functional studies of synIX. L.H.M. and W.Z. developed and evaluated the chromosome substitution method. L.H.M. A.L.H. J.C. V.F. E.L. and G.S. performed sequencing and data analyses. L.H.M. W.Z. and J.D.B. wrote the manuscript. L.A.M. H.J. J.L. M.S.T. and W.R.B. provided advice. S.C. J.D.B. J.S.B. and L.M.S. provided lab supervision. All authors reviewed and edited the manuscript. J.D.B. is a founder and director of CDI Labs, Inc. a founder of and consultant to Neochromosome, Inc, a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the scientific advisory board of the following: Logomix, Inc. Sangamo, Inc. Modern Meadow, Inc. Rome Therapeutics, Inc. Sample6, Inc. Tessera Therapeutics, Inc. and the Wyss Institute. J.S.B. is a founder of Neochromosome, Inc. consultant to Opentrons Labworks, Inc. and advisor to Reflexion Pharmaceuticals, Inc. Publisher Copyright: © 2023 The Authors

PY - 2023/11/8

Y1 - 2023/11/8

N2 - 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.

AB - 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.

U2 - 10.1016/j.xgen.2023.100419

DO - 10.1016/j.xgen.2023.100419

M3 - Article

C2 - 38020974

SN - 2666-979X

VL - 3

JO - Cell Genomics

JF - Cell Genomics

IS - 11

M1 - 100419

ER -

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

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