Letter | Published:

Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast

Naturevolume 560pages392396 (2018) | Download Citation

Abstract

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere–telomere fusions and genome duplication events3,4,5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7,8,9,10, but CRISPR–Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR–Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome–chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.

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References

  1. 1.

    Kandul, N. P., Lukhtanov, V. A. & Pierce, N. E. Karyotypic diversity and speciation in Agrodiaetus butterflies. Evolution 61, 546–559 (2007).

  2. 2.

    Taylor, R. W. Ants with Attitude: Australian Jack-jumpers of the Myrmecia pilosula species complex, with descriptions of four new species (Hymenoptera: Formicidae: Myrmeciinae). Zootaxa 3911, 493–520 (2015).

  3. 3.

    Gordon, J. L., Byrne, K. P. & Wolfe, K. H. Mechanisms of chromosome number evolution in yeast. PLoS Genet. 7, e1002190 (2011).

  4. 4.

    Kellis, M., Birren, B. W. & Lander, E. S. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428, 617–624 (2004).

  5. 5.

    Wolfe, K. H. & Shields, D. C. Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708–713 (1997).

  6. 6.

    IJdo, J. W., Baldini, A., Ward, D. C., Reeders, S. T. & Wells, R. A.; JW. Origin of human chromosome 2: an ancestral telomere-telomere fusion. Proc. Natl Acad. Sci. USA 88, 9051–9055 (1991).

  7. 7.

    Ueda, Y. et al. Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes. J. Biosci. Bioeng. 113, 675–682 (2012).

  8. 8.

    Neurohr, G. et al. A midzone-based ruler adjusts chromosome compaction to anaphase spindle length. Science 332, 465–468 (2011).

  9. 9.

    Titos, I., Ivanova, T. & Mendoza, M. Chromosome length and perinuclear attachment constrain resolution of DNA intertwines. J. Cell Biol. 206, 719–733 (2014).

  10. 10.

    DiCarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR–Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

  11. 11.

    Schubert, I. & Oud, J. L. There is an upper limit of chromosome size for normal development of an organism. Cell 88, 515–520 (1997).

  12. 12.

    Verdaasdonk, J. S. & Bloom, K. Centromeres: unique chromatin structures that drive chromosome segregation. Nat. Rev. Mol. Cell Biol. 12, 320–332 (2011).

  13. 13.

    Winey, M. & Bloom, K. Mitotic spindle form and function. Genetics 190, 1197–1224 (2012).

  14. 14.

    Shao, Y. et al. Creating a functional single chromosome yeast. Nature https://doi.org/10.1038/s41586-018-0382-x (2018).

  15. 15.

    Lynch, M. et al. A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc. Natl Acad. Sci. USA 105, 9272–9277 (2008).

  16. 16.

    Wang, Y., Shirogane, T., Liu, D., Harper, J. W. & Elledge, S. J. Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling. Cell 112, 697–709 (2003).

  17. 17.

    Baack, E., Melo, M. C., Rieseberg, L. H. & Ortiz-Barrientos, D. The origins of reproductive isolation in plants. New Phytol. 207, 968–984 (2015).

  18. 18.

    Hou, J., Friedrich, A., de Montigny, J. & Schacherer, J. Chromosomal rearrangements as a major mechanism in the onset of reproductive isolation in Saccharomyces cerevisiae. Curr. Biol. 24, 1153–1159 (2014).

  19. 19.

    Delneri, D. et al. Engineering evolution to study speciation in yeasts. Nature 422, 68–72 (2003).

  20. 20.

    Liti, G., Barton, D. B. & Louis, E. J. Sequence diversity, reproductive isolation and species concepts in Saccharomyces. Genetics 174, 839–850 (2006).

  21. 21.

    Leducq, J. B. et al. Speciation driven by hybridization and chromosomal plasticity in a wild yeast. Nat. Microbiol. 1, 15003 (2016).

  22. 22.

    Haber, J. E., Thorburn, P. C. & Rogers, D. Meiotic and mitotic behavior of dicentric chromosomes in Saccharomyces cerevisiae. Genetics 106, 185–205 (1984).

  23. 23.

    Yona, A. H. et al. Chromosomal duplication is a transient evolutionary solution to stress. Proc. Natl Acad. Sci. USA 109, 21010–21015 (2012).

  24. 24.

    Sheltzer, J. M. et al. Aneuploidy drives genomic instability in yeast. Science 333, 1026–1030 (2011).

  25. 25.

    Bonney, M. E., Moriya, H. & Amon, A. Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes. Genes Dev. 29, 898–903 (2015).

  26. 26.

    Brown, C. A., Murray, A. W. & Verstrepen, K. J. Rapid expansion and functional divergence of subtelomeric gene families in yeasts. Curr. Biol. 20, 895–903 (2010).

  27. 27.

    Ai, W., Bertram, P. G., Tsang, C. K., Chan, T. F. & Zheng, X. F. Regulation of subtelomeric silencing during stress response. Mol. Cell 10, 1295–1305 (2002).

  28. 28.

    Mayr, E. Systematics and the Origin of Species (Columbia Univ. Press, New York, 1942).

  29. 29.

    Dobzhansky, T. Genetics and the Origin of Species (Columbia Univ. Press, New York, 1937).

  30. 30.

    De Queiroz, K. Species concepts and species delimitation. Syst. Biol. 56, 879–886 (2007).

  31. 31.

    Neta Agmon, J. T. et al. Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node. Preprint at https://www.biorxiv.org/content/early/2017/06/14/147579 (2017).

  32. 32.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  33. 33.

    Yon, J. & Fried, M. Precise gene fusion by PCR. Nucleic Acids Res. 17, 4895 (1989).

  34. 34.

    Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M. & Heyneker, H. L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).

  35. 35.

    Richardson, S. M., Wheelan, S. J., Yarrington, R. M. & Boeke, J. D. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res. 16, 550–556 (2006).

  36. 36.

    Mitchell, L. A. et al. Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science 355, eaaf4831 (2017).

  37. 37.

    Breslow, D. K. et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat. Methods 5, 711–718 (2008).

  38. 38.

    Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protocols 7, 562–578 (2012).

  39. 39.

    Wu, Y. et al. Bug mapping and fitness testing of chemically synthesized chromosome X. Science 355, eaaf4706 (2017).

  40. 40.

    Ellahi, A., Thurtle, D. M. & Rine, J. The chromatin and transcriptional landscape of native Saccharomyces cerevisiae telomeres and subtelomeric domains. Genetics 200, 505–521 (2015).

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Acknowledgements

We thank L. A. Mitchell, N. Agmon, and D. M. Truong for discussion and comments on this manuscript; L. A. Vale Silva and A. Hochwagen for sharing strains, advice and reagents; The NYU Langone Health Genome Technology Center, especially A. Heguy and P. Zappile, for deep sequencing libraries; M. S. Hogan and M. T. Maurano for help with the NextSeq 500 sequencer; Z. Kuang, X. Wang and Z. Tang for advice on bioinformatics analysis; and M. Delarue and L. Holt for help and access to spinning disk confocal microscopy. This work was supported by NSF grant MCB-1616111 to J.D.B.

Reviewer information

Nature thanks G. Liti, K. Wolfe and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Institute for Systems Genetics, NYU Langone Health, New York, NY, USA

    • Jingchuan Luo
    • , Xiaoji Sun
    •  & Jef D. Boeke
  2. Department of Molecular Biology & Genetics, JHU School of Medicine, Baltimore, MD, USA

    • Jingchuan Luo
    •  & Brendan P. Cormack

Authors

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  2. Search for Xiaoji Sun in:

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Contributions

J.L., B.P.C. and J.D.B conceived the project; J.L. and J.D.B. designed experiments, analysed results and wrote the manuscript; J.L. performed experiments; X.S. performed bioinformatics analysis; all authors read and commented on the manuscript.

Competing interests

J.D.B is a founder and Director of Neochromosome, Inc., the Center of Excellence for Engineering Biology, and CDI Labs, Inc. and serves on the Scientific Advisory Board of Modern Meadow, Inc., Recombinetics, Inc., and Sample6, Inc. All other authors declare no competing interests.

Corresponding author

Correspondence to Jef D. Boeke.

Extended data figures and tables

  1. Extended Data Fig. 1 Fusion chromosome paths and characterization.

    a, PCR verification of fusion chromosomes. Two pairs of primers are used to verify the fusion of two chromosomes, here presenting the fusion of chromosomes I and III. Only with successful fusion would an amplicon show in fusion chromosome junction PCR. In addition, the centromere region PCR amplicon is shorter, owing to the deletion of a centromere. Marker: 2-log DNA marker. b, Circos diagrams for different values of n are shown in the upper panel, with 16 chromosomes laid out in circles in a clockwise manner. Each grey line connecting each pair of chromosomes represents a possible intertelomeric link. The dashed coloured lines represent the path we chose. (Open circle indicates the starting chromosome, arrows show direction of fusion chromosomes.) Each fusion chromosome is labelled in the same colour, providing detailed information on fusion chromosome paths. The underlined chromosome has the active centromere for the fusion chromosome. Unchanged chromosomes are not shown, but the number of unchanged chromosome is indicated after +, for example, +9 means nine unchanged chromosomes. Fusion chromosome lengths are shown below. Please note that the length of the rDNA array (normally 1–2 Mb)is omitted here. *Chromosome containing rDNA array. In addition, telomeric ends are clearly labelled with the original chromosomes; L/R stands for left/right telomere. Active centromeres are written on the right of each fusion chromosome diagram. When we deleted CEN15 and made 4 bp of mutations in gRNA sites in BY4741 to fuse chromosomes X and XV together, the fusion strain grew more slowly than the wild type. The growth defect could be due to altered expression of the gene neighbouring CEN15 (YOR001W; RRP6). For this reason, CEN15 was maintained in the remaining centromere. c, Fusion chromosome paths from n = 4 to n = 2. Red lettering indicates the chromosome that has an active centromere in the compound chromosome. d, Multiple strategies that we attempted to construct an n = 1 strain. We tried changing chromosome arm length (strategy 3), centromere position (strategy 2), and which telomeres remained (strategy 1 and 2). In strategy 2, we attempted both versions of n = 1 strains, keeping either CEN7 or CEN15 as the active centromere. In addition, we also attempted the strategy 1 in both SIR2+ and sir2Δ backgrounds.

  2. Extended Data Fig. 2 Growth fitness assays.

    a, Serial dilution assays in seven different conditions with n = 16 through n = 4 strains. b, Growth curve for BY4741, the n = 4 strain and the n = 2 strain in YPD medium at 30 °C. c, Doubling time calculation for these three strains. Each experimental group was tested in biological quadruplicate. Source data

  3. Extended Data Fig. 3 Whole-genome coverage maps and SNPs comparison.

    a, Whole-genome coverage map of BY4741. Chromosomes are ranged according to their size. x-axis shows chromosome coordinate, and y-axis shows the coverage of reads that map to the reference genome of S288C. b, Whole-genome coverage map of n = 2 strain. For comparison, we mapped the reads back to the S288C genome. The main difference is telomere reads. Owing to deletion of telomeres in n = 2, either no reads or only few reads align back to fused telomere ends. c, Each round of CRISPR–Cas9 experiments takes at least 70 generations. The nucleotide mutation rate is 0.33 × 10−9 per base per generation15. If we multiply the number of rounds of CRISPR–Cas9 (16 – n) × 70 generations × yeast genome size (bp) 12 × 106 × 0.33 × 10−9, it gives the expected number of SNPs, as indicated in the table.

  4. Extended Data Fig. 4 Chromosome plots of gene expression change in n = 2 vs n = 16 strains.

    y-axis, log2 (fold change of gene expression (n = 2/n = 16)); x-axis, chromosome coordinates. Red arrows point to the four remaining telomeres in n = 2 strains. Source data

  5. Extended Data Fig. 5 DAPI staining of nucleus and sporulation efficiency for diploids.

    a, DAPI staining of nuclei in diploids. b, Sporulation efficiencies for 2n = 32, 2n = 16, 2n = 8 and 2n = 4 homotypic diploids. y-axis: percentage of asci that have 0–4 spores. x-axis: different diploid strains. Sporulation efficiency was measured after shifting diploid strains in the sporulation medium for 10 days in room temperature. c, Fractions of asci with 0–4 viable spores in heterotypic and homotypic crosses. Source data

  6. Extended Data Table 1 gRNA sequences and fusion efficiency of each fusion step
  7. Extended Data Table 2 Summary table of fusion chromosome strains
  8. Extended Data Table 3 Variants identified from genome sequencing of different n strains
  9. Extended Data Table 4 Gene expression changes in n = 2, 4 and 8 strains
  10. Extended Data Table 5 Primers for PCR verification of fusion chromosomes

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https://doi.org/10.1038/s41586-018-0374-x

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