Creating functional chromosome fusions in yeast with CRISPR–Cas9

Abstract

CRISPR–Cas9-facilitated functional chromosome fusion allows the generation of a series of yeast strains with progressively reduced chromosome numbers that are valuable resources for the study of fundamental concepts in chromosome biology, including replication, recombination and segregation. We created a new yeast strain with a single chromosome by using the protocol for chromosome fusion described herein. To ensure the accuracy of chromosome fusions in yeast, the long redundant repetitive sequences near linear chromosomal ends are deleted, and the fusion orders are correspondingly determined. Possible influence on gene expression is minimized to retain gene functionality. This protocol provides experimentally derived guidelines for the generation of functional chromosome fusions in yeast, especially for the deletion of repetitive sequences, the determination of the fusion order and cleavage sites, and primary evaluation of the functionality of chromosome fusions. Beginning with design, one round of typical chromosome fusion and functional verifications can be accomplished within 18 d.

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Fig. 1: Overview of the generation of a functional single-chromosome yeast strain.
Fig. 2: Repetitive sequences near the chromosome ends.
Fig. 3: Diagram of different options for deletion of telomere-associated RSs and determination of the order of chromosome fusion.
Fig. 4: Different trial experiments for the fusion of chromosomes XV and XI.
Fig. 5: Growth curves of yeast strains with pairwise chromosome fusions.
Fig. 6: Timeline and overview of one round of chromosome fusion.
Fig. 7: Schematic representation of the design and construction of the gRNA expression plasmid.
Fig. 8: Design and construction of donor DNA cassettes for chromosome fusion.
Fig. 9: Confirmation of chromosome fusion.
Fig. 10: Schematic diagram of the removal of the selection marker and gRNA expression plasmid.

Data availability

The plasmids used in this protocol, including pCas9 (accession number 1.2624), pHIS426 (accession number 1.2623) and pXX11 (accession number 1.2613), can be obtained from the Registry and Database of Bioparts for Synthetic Biology (http://npbiosys.scbit.org/strainOrder) upon reasonable request. All relevant data are reported in the article.

References

  1. 1.

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

  2. 2.

    McClintock, B. The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234–282 (1941).

  3. 3.

    Hill, A. & Bloom, K. Genetic manipulation of centromere function. Mol. Cell. Biol. 7, 2397–2405 (1987).

  4. 4.

    Hill, A. & Bloom, K. Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 1368–1370 (1989).

  5. 5.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  6. 6.

    Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  7. 7.

    Shao, Y. et al. Creating a functional single-chromosome yeast. Nature 560, 331–335 (2018).

  8. 8.

    Goffeau, A. et al. Life with 6000 genes. Science 274, 546–567 (1996).

  9. 9.

    Qin, Z. & Cohen, S. N. Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing. Genes Dev. 14, 1789–1796 (2000).

  10. 10.

    Akgun, E. et al. Palindrome resolution and recombination in the mammalian germ line. Mol. Cell. Biol. 17, 5559–5570 (1997).

  11. 11.

    Leach, D. R. Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair. BioEssays 16, 893–900 (1994).

  12. 12.

    Henikoff, S. & Henikoff, J. G. “Point” centromeres of Saccharomyces harbor single centromere-specific nucleosomes. Genetics 190, 1575–1577 (2012).

  13. 13.

    Jager, D. & Philippsen, P. Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion. EMBO J. 8, 247–254 (1989).

  14. 14.

    Pobiega, S. & Marcand, S. Dicentric breakage at telomere fusions. Genes Dev. 24, 720–733 (2010).

  15. 15.

    Lopez, V. et al. Cytokinesis breaks dicentric chromosomes preferentially at pericentromeric regions and telomere fusions. Genes Dev. 29, 322–336 (2015).

  16. 16.

    Luo, J., Sun, X., Cormack, B. P. & Boeke, J. D. Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast. Nature 560, 392–396 (2018).

  17. 17.

    Shao, Y., Lu, N., Qin, Z. & Xue, X. CRISPR–Cas9 facilitated multiple-chromosome fusion in Saccharomyces cerevisiae. ACS Synth. Biol 7, 2706–2708 (2018).

  18. 18.

    Xue, X. et al. MEGA (multiple essential genes assembling) deletion and replacement method for genome reduction in Escherichia coli. ACS Synth. Biol. 4, 700–706 (2015).

  19. 19.

    Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

  20. 20.

    Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172–2175 (1989).

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Acknowledgements

This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000, Z.Q.; 153D31KYSB20160074, Z.Q.), the National Natural Science Foundation of China (31830105, Z.Q.; 31770099 and 31200059, X.X.), Shanghai Research Project (18JC1420200, Z.Q.) and China Postdoctoral Science Foundation (2018M640427 and 2019T120362, Y.S.)

Author information

Z.Q. and X.X. designed and analyzed all the experiments. Y.S. constructed the chromosome fusion yeast strains and performed PCR verification. N.L. conducted the PFGE confirmation experiment and growth assays. X.X. and Y.S. wrote the primary manuscript with a substantial contribution from Z.Q.

Correspondence to Xiaoli Xue or Zhongjun Qin.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Protocols thanks Meru Sadhu and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Shao, Y. et al. Nature 560, 331–335 (2018): https://doi.org/10.1038/s41586-018-0382-x

Shao, Y., Lu, N., Qin, Z. & Xue, X. ACS Synth. Biol. 7, 2706–2708 (2018): https://doi.org/10.1021/acssynbio.8b00397

Shao, Y. et al. Cell Res. 29, 87–89 (2019): https://doi.org/10.1038/s41422-018-0110-y

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