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A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica.

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Title: A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica.
Authors: Zhiyong Cui1, Huihui Zheng1, Jinhong Zhang1, Zhennan Jiang1, Ziwei Zhu1, Xiaoqin Liu1, Qingsheng Qi1,2 qiqingsheng@sdu.edu.cn, Jin Hou1 houjin@sdu.edu.cn
Source: Applied & Environmental Microbiology. Mar2021, Vol. 87 Issue 6, p1-16. 16p.
Subjects: DNA repair, Double-strand DNA breaks, Genome editing, Genomes, Genes, CRISPRs
Abstract: 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 G1 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 homologyindependent 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 multiplegene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes. [ABSTRACT FROM AUTHOR]
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Abstract: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 G1 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 homologyindependent 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 multiplegene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes. [ABSTRACT FROM AUTHOR]
ISSN:00992240
DOI:10.1128/AEM.02666-20