Development of a CRISPR/Cas9-mediated homology-independent tool for targeted genome integration in Yarrowia lipolytica.

Yarrowia lipolytica has been extensively used for producing essential chemicals and enzymes. Like most other eukaryotes, non-homologous 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 optimizing the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, cell cycle, and the integration sites. Thus, targeted integration rate reached to 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes 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 8417 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 iteratively integrate of multiple genes pathway in Y. lipolytica. This tool may serve as a potential supplement for current HR-dependent genome editing methods for eukaryotes.