A Lambda Red Recombinase‐Assisted CRISPR /Cas9 System for Efficient and Precise Genome Editing in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a powerful delivery tool for plant genetic transformation and a pivotal platform for agricultural biotechnology. However, laboratory strains of Agrobacterium have remained genetically unoptimized for decades, with genome engineering hindered by inefficient, endogenous homologous recombination systems. Here, we developed and optimized a lambda (λ) Red recombinase‐assisted CRISPR/Cas9 system that enables efficient and precise introduction of point mutations, insertions, deletions and large fragment replacements into both the chromosome and Ti plasmids of different A. tumefaciens strains, with minimal off‐target editing. Editing efficiencies across eighteen target sites ranged from 26.7% to 100%, demonstrating the versatility of this method. Furthermore, we incorporated a SacB counter‐selection marker into our system that facilitates the plasmids curing after editing. Using this platform, we sequentially deleted four antibiotic‐resistance loci in strain EHA105, generating an antibiotic‐sensitive derivative, EHA105‐S, that maintains wild‐type growth kinetics and transformation efficiency. Additionally, we engineered the microbe‐associated molecular pattern (MAMPs) of two elongation factor Tu (EF‐Tu) genes, allowing the A. tumefaciens to escape EF‐Tu receptor (EFR) perception. The resulting engineered strains displayed enhanced transient expression efficiency in Arabidopsis thaliana . Our λ Red‐assisted CRISPR/Cas9 system thus provides a robust, scalable and user‐friendly tool for Agrobacterium genome engineering, paving the way for the design of next‐generation strains to revolutionize plant transformation and advance synthetic biology applications in plants.