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

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, engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold vs. wild-type attB) and 35.6% in rice protoplasts (4.4-fold vs. 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 CAR cassette and 25% for an OTC expression cassette in human cells. In rice, via an all-in-one delivery strategy enabled precise integration of a 5.8-kb herbicide-resistance gene, yielding 24% stable integration in regenerated transgenic 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 also confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing. （NGS data to assist the att insertion efficiency and integration efficiency of recombinase in human cells and rice protoplasts; Tn5-PCR data to assist the whole-genome specificity of Bxb1_R2Q/attB(V111) gene editing tool in rice ; You need to download all split archive files. After extraction, a total of 693 data files will be obtained. The file names and their corresponding MD5 checksums are listed in the SampleReadMe.txt file.