Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions |
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| Institution: | 1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, PR China;2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai, 200237, PR China;1. Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-909, RJ, Brazil;2. Biochemical Engineering Department, School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-909, RJ, Brazil;3. Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China;2. College of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China;3. State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China;4. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China;1. McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 East Dean Keeton Street, Austin, TX 78712, USA;2. Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA;1. School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China;2. Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou 318000, China |
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| Abstract: | Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica. |
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| Keywords: | tRNA-sgRNA fusion CRISPR-Cas9 Yarrowia lipolytica Knock out/in Genome editing Secondary structure |
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