Efficient targeted T‐DNA integration for gene activation and male germline‐specific gene tagging in Arabidopsis

SUMMARY Site‐specific DNA integration is an important tool in plant genetic engineering. Traditionally, this process relies on homologous recombination (HR), which is known for its low efficiency in plant cells. In contrast, Agrobacterium ‐mediated T‐DNA integration is highly efficient for plant transformation. However, T‐DNA is typically inserted randomly into double‐strand breaks within the plant genome via the non‐homologous end‐joining (NHEJ) DNA repair pathway. In this study, we developed an approach of CRISPR/Cas9‐mediated targeted T‐DNA integration in Arabidopsis, which was more rapid and efficient than the HR‐mediated method. This targeted T‐DNA integration aided in gene activation and male germline‐specific gene tagging. Gene activation was accomplished by positioning the CaMV35S promoter at the left border of T‐DNA, thereby activating specific downstream genes. The activation of FT and MYB26 significantly increased their transcriptional expression, which resulted in early flowering and an altered pattern of cell wall thickening in the anther endothelium, respectively. Male germline‐specific gene tagging incorporates two reporters, namely, NeoR and MGH3::mCherry , within the T‐DNA. This design facilitates the creation of insertional mutants, simplifies the genetic analysis of mutated alleles, and allows for cellular tracking of male germline cells during fertilization. We successfully applied this system to target the male germline‐specific gene GEX2. In conclusion, our results demonstrated that site‐specific integration of DNA fragments in the plant genome can be rapidly and efficiently achieved through the NHEJ pathway, making this approach broadly applicable in various contexts.