Optimization of IS621 recombinase/bridge RNA-directed recombination for precise insertion of large DNA fragments in human cells

Therapeutic integration of large DNA fragments into precise genomic loci of human cells remains challenging. The IS621 recombinase, derived from the IS110 family, utilizes a bridge RNA (bRNA) containing an independently programmable target-binding loop (TBL) and donor-binding loop (DBL) to mediate sequence-specific recombination between target and donor DNA in prokaryotes. To expand its application to human cells, we rationally engineer the IS621 protein and its cognate bRNA. This ultimately leads to the development of enIS621-tebRNA, which is capable of driving site-specific, scarless insertion of large DNA fragments in human cells. We systematically evaluate the insertion efficiency of this system across multiple cell types, achieving integration rates up to 27.75% for kilobase-scale DNA cargos. Furthermore, we successfully achieve functional insertion of a CD19 chimeric antigen receptor (CAR) at relevant loci for T cell therapy, and establish site-specific integration of the gene encoding human Factor IX with implications for hemophilia B gene therapy. These results highlight the potential of the engineered enIS621-tebRNA system as a versatile platform for precise insertion of large DNA fragments in human cells. Large DNA insertion at precise genomic sites remains challenging in human cells. Here, authors engineer the IS621 recombinase and bridge RNA (enIS621- tebRNA) to enable scarless insertion of large DNA fragments, including therapeutic cargos for CAR-T cells and for future hemophilia B applications.