Directed evolution on compact landing pads yields highly efficient recombinases for large DNA integration.

Site-specific DNA recombinases are powerful tools for genome engineering. The recent convergence of DNA recombinase-mediated technology with prime editing has established a promising new frontier for large-scale genomic integration. However, this strategy has limitations, including low intrinsic catalytic efficiency of DNA recombination enzymes and inefficiency of their long DNA landing pad insertions. To overcome these challenges, we developed a novel directed evolution strategy using progressively shortening landing pads as a selective pressure. The resulting evolved variants, VK and AVK, exhibited substantially enhanced intrinsic activity that is maintained on short-length landing pads. This enables a powerful synergistic effect with increased insertion efficiency of prime editing, leading to a dramatic increase in overall integration efficiency while maintaining high genomic specificity. This work thus provides a new class of highly efficient recombinases and a robust engineering strategy with broad applicability for both the development of gene therapies and fundamental research.