Site-specific DNA insertion into the human genome with engineered recombinases.

Engineered large serine recombinases achieve up to 53% integration efficiency with 97% genome-wide specificity at an endogenous human locus for inserting up to 12 kb.

• Why it matters: Large DNA insertions are needed for research and therapeutic gene and cell applications, but existing LSRs have low on-target rates and high off-target activity.
• What they did: The team used directed evolution, structural analysis, and computational models to find additive mutations, then optimized donor DNA and used dCas9 fusions for coordinated target and donor recruitment.
• The result: Top variants (superDn29-dCas9, goldDn29-dCas9, hifiDn29-dCas9) enabled stable, single-step integration of large DNA cargoes up to 12 kb in non-dividing cells, stem cells, and primary human T cells.

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