Engineered genomic attachment sites for site-specific recombinases enable high-efficiency integration in plants and human cells.

Here we developed a DNA-centric strategy for optimizing site-specific recombination by rationally engineering chimeric attachment sites. The high-activity att variants enhance Bxb1-mediated integration efficiency in human cells and plants. Among these att variants, the engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold versus wild-type attB) and 35.6% in rice protoplasts (4.4-fold versus wild-type attB). When paired with an engineered single protein mutant in the Bxb1 catalytic domain, the optimized system achieved targeted integration efficiencies of 31% for a CD19 chimeric antigen receptor cassette and 25% for an ornithine transcarbamylase expression cassette in human cells. In rice, these engineered variants enabled integration of a 5.8 kb herbicide-resistance cassette at a targeted genomic locus, with stable integration detected in 24% of regenerated plants. Oxford Nanopore-based long-read sequencing of edited plants reveals complete and precise insertion with high specificity. Propagation of edited seedlings to T1 plants confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing.