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 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 Through optimization of the cleavage efficiency of Cas9, targeted integration of a 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 This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in 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 This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.