title: Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.
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.
authors.affiliation: School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Shuliang
authors.initials: S
authors.lastname: Gao
authors.affiliation: College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai, 200234, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Tong
authors.affiliation: 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.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wen
authors.affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Ge
authors.affiliation: School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. hccb001@163.com.|Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai, 200040, China. hccb001@163.com.
authors.forename: Daijie
authors.initials: D
authors.lastname: Chen
authors.affiliation: 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. yjiang01@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai, 201201, China. yjiang01@sibs.ac.cn.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: 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. syang@sibs.ac.cn.|Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai, 201201, China. syang@sibs.ac.cn.|Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing, 211816, China. syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair; D000072669:Gene Editing; D005800:Genes, Fungal; D016681:Genome, Fungal; D059767:Recombinational DNA Repair; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism
keywords: CRISPR; Genome-editing; Yarrowia lipolytica