Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 87, Issue 6, Pages -Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.02666-20
Keywords
Yarrowia lipolytica; CRISPR/Cas9; nonhomologous end joining; targeted genome integration tool
Categories
Funding
- National Key R&D Program of China [2019YFA0904900]
- National Natural Science Foundation of China [31730003, 31961133014]
- National Defense Science and Technology Innovation Zone Foundation of China
- China Postdoctoral Science Foundation-funded project [2019M662340]
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This study developed a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. The tool does not require homologous templates, and can be used to rapidly verify genetic elements and iteratively integrate multiple-gene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.
Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica. Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica. Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G, phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolytica. IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.
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