Research Articles

“Perfect” designer chromosome V and behavior of a ring derivative

See allHide authors and affiliations

Science  10 Mar 2017:
Vol. 355, Issue 6329, eaaf4704
DOI: 10.1126/science.aaf4704
  • Synthesis, cyclization, and characterization of synV.

    (A) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. (B) CRISPR/Cas9 strategy for multiplex repair. (C) Colonies of wtV, synV, and ring_synV strains.

  • Fig. 1 Characterization of synV.

    (A) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs) (fig. S1A). (B) Phenotypic analysis of synV (yXZX347, yeast_chr05_9_01) and wtV (BY4741) strains under different conditions (figs. S3 and S6). (C) PCRTag analysis (one PCRTag per ~30 kb) of synV (yXZX512, version “yeast_chr05_9_04”) (figs. S8 and S9). (D) Transcriptome analysis of synV (yXZX347, yeast_chr05_9_01) relative to wild-type strain (BY4741). Genes with significant expression changes are labeled in red. YER187W and YER188W are telomere adjacent and likely reduced in expression because of removal of subtelomeric DNA. YPD, yeast extract, peptone, and dextrose; SC, synthetic complete medium; SD, synthetic dextrose; YPG, yeast extract peptone glycerol; YPE, yeast extract peptone ethanol.

  • Fig. 2 Schematic outlining cotransformation strategy to correct synV strain to design.

    (A) Representative variant regions encoded by initial synV isolate (yXZX347, yeast_chr05_9_01) were targeted for correction by the methods indicated in (B) to (E). A detailed map of variants is provided in fig. S12. (B) Multiplex variant repair by means of integrative cotransformation strategy. 13-2, half part of variant region 13; n, last variant region. (C) An example of successfully corrected variants by means of integrative cotransformation strategy. A tRNA gene and intron were replaced with a loxPsym site through a single cotransformation procedure; target region “13-2” was repaired simultaneously. (D) Multiplex variant repair by CRISPR/Cas9 strategy. n, last variant region. (E) An example of successfully corrected variants by means of CRISPR/Cas9 strategy. For “14,” a single mutation was repaired with the CRISPR/Cas9 method, and the region “16” was corrected simultaneously (fig. S12 and tables S1 and S3).

  • Fig. 3 Construction, characterization, and meiotic stability analysis of ring_synV chromosome.

    (A) Circularization of synV (yXZX538, yeast_chr05_9_05) yielded a version of ring_synV (yXZX565) with gene content identical to linear synV. (B) PCR verification of ring_synV and ring_wtV. (C) PFGE of ring_synV chromosome and stability verification. Linear synV (yXZX538, yeast_chr05_9_05, red triangle) migrates faster than native V (wtV), and the circular chromosome ring_synV is not detected. After 5 days of subculturing, the diploid strain ring_synV/ring_synV (yXZX598) was analyzed with PFGE, and no linear synV appeared. (D) Phenotypic analysis of synV and ring_synV on various media (fig. S13). (E) Colonies of synV and ring_synV on various media. (F) Evaluation of spore viability from 50 tetrads of diploid strains harboring the indicated wtV and synV linear and ring derivatives.

Supplementary Materials

  • "Perfect" designer chromosome V and behavior of a ring derivative

    Ze-Xiong Xie, Bing-Zhi Li, Leslie A. Mitchell, Yi Wu, Xin Qi, Zhu Jin, Bin Jia, Xia Wang, Bo-Xuan Zeng, Hui-Min Liu, Xiao-Le Wu, Qi Feng, Wen-Zheng Zhang, Wei Liu, Ming-Zhu Ding, Xia Li, Guang-Rong Zhao, Jian-Jun Qiao, Jing-Sheng Cheng, Meng Zhao, Zheng Kuang, Xuya Wang, J. Andrew Martin, Giovanni Stracquadanio, Kun Yang, Xue Bai, Juan Zhao, Meng-Long Hu, Qiu-Hui Lin, Wen-Qian Zhang, Ming-Hua Shen, Si Chen, Wan Su, En-Xu Wang, Rui Guo, Fang Zhai, Xue-Jiao Guo, Hao-Xing Du, Jia-Qing Zhu, Tian-Qing Song, Jun-Jun Dai, Fei-Fei Li, Guo-Zhen Jiang, Shi-Lei Han, Shi-Yang Liu, Zhi-Chao Yu, Xiao-Na Yang, Ken Chen, Cheng Hu, Da-Shuai Li, Nan Jia, Yue Liu, Lin-Ting Wang, Su Wang, Xiao-Tong Wei, Mei-Qing Fu, Lan-Meng Qu, Si-Yu Xin, Ting Liu, Kai-Ren Tian, Xue-Nan Li, Jin-Hua Zhang, Li-Xiang Song, Jin-Gui Liu, Jia-Fei Lv, Hang Xu, Ran Tao, Yan Wang, Ting-Ting Zhang, Ye-Xuan Deng, Yi-Ran Wang, Ting Li, Guang-Xin Ye, Xiao-Ran Xu, Zheng-Bao Xia, Wei Zhang, Shi-Lan Yang, Yi-Lin Liu, Wen-Qi Ding, Zhen-Ning Liu, Jun-Qi Zhu, Ning-Zhi Liu, Roy Walker, Yisha Luo, Yun Wang, Yue Shen, Huanming Yang, Yizhi Cai, Ping-Sheng Ma, Chun-Ting Zhang, Joel S. Bader, Jef D. Boeke, Ying-Jin Yuan

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Figs. S1 to S14
    • Tables S1 to S7

Navigate This Article