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

Insertions of large DNA sequences into the genome are broadly enabling for research and therapeutic applications. Large serine recombinases (LSRs) can mediate direct, site-specific genomic integration of multi-kilobase DNA sequences without a pre-installed landing pad, albeit with low insertion rates and high off-target activity. Here we present an engineering roadmap for jointly optimizing their DNA recombination efficiency and specificity. We combine directed evolution, structural analysis and computational models to rapidly identify additive mutational combinations. We further enhance performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Our top engineered LSR variants, superDn29-dCas9, goldDn29-dCas9 and hifiDn29-dCas9, achieve up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus and effectively integrate large DNA cargoes up to 12 kb for stable expression in non-dividing cells, stem cells and primary human T cells. Rational engineering of DNA recombinases enables precise and efficient single-step genome insertion for diverse applications across gene and cell therapies.