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Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

  • Biotechnology Methods
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

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Acknowledgments

We would like to thank Professor Hal S. Alper for the generous gift of plasmid pMCSCen1. This study was financed by the Ministry of Science and Technology of China (973:2012CB721105; 863:2012AA02A704). This study was also supported in part by project (973:2014CB745101) and the STS project from CAS (KFJ-EW-STS-030).

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Authors and Affiliations

  1. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Shuliang Gao & Daijie Chen

  2. College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai, 200234, China

    Yangyang Tong

  3. CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China

    Zhiqiang Wen, Yu Jiang & Sheng Yang

  4. Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai, 200240, China

    Li Zhu & Mei Ge

  5. Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai, 200040, China

    Daijie Chen

  6. Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai, 201201, China

    Yu Jiang & Sheng Yang

  7. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing, 211816, China

    Sheng Yang

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  1. Shuliang Gao
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Correspondence to Daijie Chen, Yu Jiang or Sheng Yang.

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Gao, S., Tong, Y., Wen, Z. et al. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. J Ind Microbiol Biotechnol 43, 1085–1093 (2016). https://doi.org/10.1007/s10295-016-1789-8

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  • DOI: https://doi.org/10.1007/s10295-016-1789-8

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