Modular and efficient assembly of CRISPR vectors and construction of an apple gene editing efficiency evaluation system

【Objective】The CRISPR/Cas gene editing technology has demonstrated a broad application prospect for diverse applications in genetic improvement programs targeting apple (Malus domestica) and other perennial fruit crops. However, conventional gene editing systems exhibit critical limitations, including low efficiency vector assembly and inflexible modular architectures. We aim to develop a modular and efficient CRISPR vector system, optimize the Agrobacterium-mediated transient delivery system in apple plantlets, and establish a corresponding evaluation system for assessing the efficiency of gene editing in apple.【Methods】The conventional vectors pUC19 (DNA cloning plasmid) and pHK2 (Dicotyledonous gene editing plasmid based on pCAMBIA1300 modification) were firstly modified by inserting a pair of type ⅡS restriction enzyme sites (such as Esp3Ⅰ or BsaⅠ etc.) into selected positions, to construct the intermediate vector pUC19-Esp3Ⅰ-Bb and the plant expression vector back-bone pHK2-BsaⅠ-Bb. Conventional plasmids were converted into modified vectors compatible with the Golden Gate cloning system respectively. Subsequently, the e35S promoter, SpyCas9 coding sequence and NOS terminator were amplified by PCR and seamlessly assembled into pUC19-Esp3Ⅰ-Bb vector to construct expression module A (e35S-Cas9- NOS); For targeted editing of apple PDS (Phytoene desaturase) gene, the specific gRNA (guide RNA) sequence was designed and constructed expression module B (AtU6-gRNA) through PCR amplification using primers containing BsaⅠ restriction enzyme site; Based on the same strategy, the RUBY reporter element was amplified by primers with additional BsaⅠ restriction enzyme site to form expression module C. These three modules (A\B\C) were assembled with the plant expression vector backbone pHK2-BsaⅠ-Bb using Golden Gate cloning technology, ultimately establishing the modular CRISPR gene editing system e35S-Cas9-AtU6-gRNA-RUBY. This platform enables flexible replacement and rapid reconstruction of core components including Cas proteins, promoters, and gRNA expression cassettes, and establishes a system for visual detection of precise plasmid transfection by integrating the RUBY reporter system.【Results】We developed a modular vector assembly platform through strategic engineering of conventional plasmids pUC19 and pHK2. By employing type ⅡS restriction enzyme sites (such as Esp3Ⅰ or BsaⅠ), we constructed two core components: the intermediate vector (pUC19-Esp3Ⅰ-Bb) and the plant expression vector backbone (pHK2-BsaⅠ-Bb). The pUC19-Esp3Ⅰ-Bb vector enabled the integration of multiple DNA fragments into a single transcriptional module, generating intermediate constructs designated as pUC19- Module A/B/C. These modules were subsequently assembled into the plant expression vector backbone pHK2-BsaⅠ-Bb through a seamless cloning strategy. This system demonstrated exceptional modular compatibility, enabling seamless integration of multiple expression modules and rapid adaptation of the vector's functional components as required. To validate this system, we constructed three core modules: a Cas9 expression cassette was generated using pUC19-Esp3Ⅰ-Bb vector (e35S-Cas9-NOS, expression Module A), a gRNA transcription unit (AtU6-gRNA, expression Module B), and a visual reporter system (RUBY, expression Module C). Hierarchical assembly of these components into the plant expression vector backbone pHK2-BsaⅠ-Bb yielded a multifunctional CRISPR gene editing plasmid (e35SCas9-AtU6-gRNA-RUBY) that concurrently encoded genome-editing machinery and a transformation marker. This system significantly improves the efficiency of vector assembly by enabling concurrent integration of CRISPR/Cas components and reporter genes into a single binary expression plasmid. Beyond providing an optimized framework for plant gene editing vector construction, this platform allows for flexible adaptation of critical components (such as Cas proteins, guide RNAs and promoters etc.) in subsequent experimental studies, thereby accommodating species specific editing requirements across diverse plant systems. Functional validation revealed that the CRISPR gene editing plasmid e35S-Cas9- AtU6-gRNA-RUBY could be transiently expressed in tobacco and showed good visual indication function. Furthermore, vacuum infiltration mediated transient transformation of apple leaves confirmed PDS gene editing through RT-PCR amplification and Sanger sequencing of target loci, and preliminarily evaluated the editing efficiency of gRNAs targeting the apple PDS gene. The experimental data demonstrated variable editing efficiencies among distinct gRNAs. Notably, gRNA variation in editing efficiency underscored the importance of pre-screening guide RNAs before stable transformation. Collectively, the vector system developed high transformation efficiency and accurate editing ability. These results establish a robust and adaptable vector platform that streamlines the construction of plant gene-editing vectors and provides a reliable toolkit for plant genome engineering.【Conclusion】The modular vector system e35S-Cas9-AtU6-gRNA-RUBY is based on Golden Gate technology, and has achieved the seam-less assembly of multiple DNA fragments in a single reaction through the synergistic action of type ⅡS restriction enzyme sites (such as Esp3Ⅰ or BsaⅠ etc.) and T4 DNA ligase. This significantly simplifies the operation process, shortens the time, and improves the efficiency and precision of the construction of gene editing vectors. This platform enables efficient and accurate seamless integration of multiple gene modules, serves as the primary vector framework for subsequent optimization of the apple gene editing system, and offers a novel solution for enhancing the efficiency and operability of apple gene editing technology. It accommodates the flexible combination of gene editing requirements and provides a universal tool and methodological framework for advancing gene editing technology in other horticultural fruit crops.