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.

DOI: 10.1007/s10295-016-1789-8

Extracted Key Phrases

7 Figures and Tables

Statistics

02004002017
Citations per Year

118 Citations

Semantic Scholar estimates that this publication has 118 citations based on the available data.

See our FAQ for additional information.

Cite this paper

@article{Gao2016MultiplexGE, title={Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system}, author={Shuliang Gao and Yangyang Tong and Zhiqiang Wen and Li Zhu and Mei Ge and Daijie Chen and Yu Jiang and Sheng Yang}, journal={Journal of Industrial Microbiology & Biotechnology}, year={2016}, volume={43}, pages={1085-1093} }