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

Technologies for precisely inserting large DNA sequences into the genome are critical for diverse 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, but current approaches suffer from low insertion rates and high off-target activity. Here, we present a comprehensive engineering roadmap for the joint optimization of DNA recombination efficiency and specificity. We combined directed evolution, structural analysis, and computational models to rapidly identify additive mutational combinations. We further enhanced performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Top engineered LSR variants achieved up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus, and effectively integrated large DNA cargoes (up to 12 kb tested) for stable expression in challenging cell types, including non-dividing cells, human embryonic stem cells, and primary human T cells. This blueprint for rational engineering of DNA recombinases enables precise genome engineering without the generation of double-stranded breaks.