A CRISPR‐Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica

ABSTRACT Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology‐directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position‐independent gene integration with predictable transcriptional output across a 12.88‐fold dynamic range. Second, we established a dual‐readout high‐throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus‐specific homologous recombination (HR) efficiency while eliminating false positives by dominant non‐homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS) 3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single‐gene integration was increased from 46.5% to 77.5% while the dual‐locus editing efficiency reached 64.1% when using 500‐bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.