Development of an endogenous promoter-driven CRISPR/Cas9 system for genome editing in Fraxinus mandshurica

CRISPR/Cas9-mediated genome editing has revolutionized tree improvement by enabling precise trait modification, accelerating breeding cycles, and enhancing forestry sustainability. Fraxinus mandshurica, valued for its desirable traits and adaptability, serves as a strategic focus for the National Reserve Forest Project (NRFP) and forestry germplasm resource breeding and quality improvement in China. Developing a species-specific genome editing system is crucial for valuable yet recalcitrant species like F. mandshurica. In this study, the development of a species-specific CRISPR/Cas9 platform is presented for F. mandshurica, which incorporates endogenous promoter engineering, sgRNA optimization, light quality modulation, and temperature control protocols to enhance genome editing efficiency. Truncated endogenous FmU6 promoter variants (FmU6-6-4 and FmU6-7-4) drove sgRNA expression at levels 3.36 and 3.11 times higher than that of the AtU6-26 promoter. The expression of Cas9 was controlled by the endogenous constitutive FmECP3 promoter, exhibiting an activity 5.48 times greater than the positive control. A highly active sgRNA4 targeting FmPDS1/2 was identified, demonstrating a cleavage efficiency of 36.10%. Heat treatment at 37 °C effectively increased the Cas9 cleavage efficiency to 7.77 times that observed at 22 °C. Chimeric albino mutants with an editing efficiency of 18.2% were obtained through transient and stable transformations, combined with light quality optimization and heat treatment during different regeneration stages. The mutation types included nucleotide insertions, deletions, and substitutions, leading to early termination codons and truncated FmPDS1/2 protein. Additionally, mutations in FmPDS1/2 resulted in albino phenotypes and a reduction in chlorophyll content to 46.44%-58.88%. This optimized system provides a robust platform for functional genomics studies and trait improvement in F. mandshurica, with potential applications in forestry biotechnology.