Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome
Matthew G. Durrant, Alison Fanton, Josh Tycko, Michaela M. Hinks, Sita S. Chandrasekaran, Nicholas T. Perry, Julia M. Schaepe, Peter P. Du, Peter Lotfy, Michael C. Bassik, Lacramioara Bintu, Ami S. Bhatt, Patrick D. Hsu
Abstract
Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.