Retargeted Serine Integrases Enable Precise Large-Gene Insertion

Serine integrases such as Bxb1 can integrate large DNA constructs with high precision but, until now, only at sites where their natural attachment sequence has already been introduced into the genome – typically via prime editing, adding a layer of complexity to therapeutic development. To overcome this, the authors used structural modelling, a single round of directed evolution and screening in human cells to identify three specificity-determining regions of Bxb1 – termed the helix, loop and hairpin – that can be re-engineered to recognise new DNA sequences.

Combining evolved variants of these regions (MINT constructs) with previously described activity-enhancing Bxb1 mutations and fused zinc-finger DNA-binding domains, the team retargeted Bxb1 to the AAVS1 safe-harbour locus and the TRAC locus, both within the T cell receptor α constant region, where wild-type Bxb1 shows no detectable activity.

In K562 cells, the optimised constructs achieved 35% targeted integration at TRAC, and genome-wide specificity assays showed strong on-target preference over candidate off-target sites. In primary human CD4⁺CD8⁺ T cells, delivery of Bxb1 variants as mRNA, alongside a nanoplasmid GFP donor, yielded up to 29% stable targeted integration 14 days after nucleofection, with minimal effects on T cell viability or phenotype.

The authors also demonstrated compatibility with AAV-mediated donor delivery and payload integration up to 12 kb, and propose that a growing archive of pre-characterised helix and hairpin variants could eventually enable in silico design of custom Bxb1 variants for new target sites without further directed evolution.

The study was led by Friedrich Fauser, Sebastian Arangundy-Franklin, Jessica E. Davis and Jeffrey C. Miller at Sangamo Therapeutics. It was published in Nature Biotechnology on 29 June 2026.