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


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Article Info

DATE

2016-06-27

AUTHORS

Shuliang Gao, Yangyang Tong, Zhiqiang Wen, Li Zhu, Mei Ge, Daijie Chen, Yu Jiang, Sheng Yang

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. More... »

PAGES

1085-1093

References to SciGraph publications

  • 2012-12-09. Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains in BIOTECHNOLOGY LETTERS
  • 2012-02-15. RNA-guided genetic silencing systems in bacteria and archaea in NATURE
  • 2013-07-21. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica in NATURE BIOTECHNOLOGY
  • 2012-05-19. Bioengineering of Oleaginous Yeast Yarrowia lipolytica for Lycopene Production in MICROBIAL CAROTENOIDS FROM FUNGI
  • 2014-09-13. One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica in BIOTECHNOLOGY LETTERS
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  • 2006-08-23. Mechanism of homologous recombination: mediators and helicases take on regulatory functions in NATURE REVIEWS MOLECULAR CELL BIOLOGY
  • 2013-02-20. Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining in CURRENT GENETICS
  • 2011-03-30. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III in NATURE
  • 1994-07. Multiple-copy integration in the yeast Yarrowia lipolytica in CURRENT GENETICS
  • 2015-05-12. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system in CELL DISCOVERY
  • 2013-10-29. The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori in CELL RESEARCH
  • 2014-10-29. Implementation of the CRISPR-Cas9 system in fission yeast in NATURE COMMUNICATIONS
  • 2014-01-20. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production in NATURE COMMUNICATIONS
  • Journal

    Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s10295-016-1789-8

    DOI

    http://dx.doi.org/10.1007/s10295-016-1789-8

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1008193065

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/27349768


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