Writing Big in Plant Genomes: Advances, Challenges and Strategies for Targeted Large-Fragment DNA Insertion.

Precise genome editing has transformed plant biology and crop improvement by enabling targeted modification of endogenous loci. Beyond gene knockout and base editing, the site-specific insertion of exogenous DNA, particularly large DNA fragments, has become a central goal for engineering complex traits, reconstructing metabolic pathways and constructing plant artificial chromosomes. A diverse toolkit is now available for targeted DNA integration, including nuclease-dependent strategies, serine and tyrosine recombinases, transposon-derived systems, and CRISPR/Cas-coupled insertion platforms. Here, we review the mechanistic principles and recent advances of these four major tools, highlighting their capacities, insertion precision and compatibility with plant systems. We compare their strengths and limitations in terms of insertion-size capacity, integration efficiency, target site flexibility and technical complexity. Emerging innovations such as AI-guided nuclease and recombinase design, fusion of Cas with recombinases or viral replication proteins and RNA-guided transposition offer promising solutions to overcome these constraints. Together, these advances are rapidly expanding the landscape of targeted DNA insertion in plants and will reinforce future applications in molecular breeding, metabolic pathway engineering and the construction of synthetic genomic architectures.