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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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.
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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.)
The authors declare no competing interests.
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.
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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Shao, Y., Lu, N., Xue, X. et al. Creating functional chromosome fusions in yeast with CRISPR–Cas9. Nat Protoc 14, 2521–2545 (2019). https://doi.org/10.1038/s41596-019-0192-0