Discovery And Validation Of Novel, High-Efficiency Large Serine Recombinases For Targeted Gene Insertion

By Avery Roberts, Kristopher Kieft, Tim Schwochert, Kyung Seo, Malik Moncalvo, Hannah Wiedner, Allie Crawley, Gavin Ellis, Matt Nethery, David Wiley, Chuck Pepe-Ranney, Amy Pooler, and Ron Chong

Large serine recombinases offer a promising route to inserting sizable genetic payloads with precision. A bioinformatics pipeline drawing from more than 10 billion protein sequences identified over 150 novel recombinase systems, approximately 80% of which demonstrated activity in mammalian cells. Testing across plasmid recombination and human genome insertion assays revealed varied integration efficiencies, including activity exceeding 80% for selected systems. The work also combines generative AI, active learning, rational design, and directed evolution to identify beneficial protein variants and improve insertion potency. In primary human T cells, a CRISPR-guided approach achieved approximately 90% landing-pad installation and 67% targeted CD19-CAR insertion at the TRAC locus across multiple donors.

See how discovery and protein-engineering strategies could expand the toolkit for programmable, large-payload gene insertion.