Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites,…

• NS
  National Science Foundation
• HH
  Howard Hughes Medical Institute
• BA
  Bill and Melinda Gates Foundation
• NI
  National Institutes of Health

  Awards: GM118062, R35 GM118062, HEK293T, U01 AI142756, HG009490, RM1 HG009490
• NH
  National Heart, Lung, and Blood Institute

  Award: R01 HL160970
• NH
  National Human Genome Research Institute

  Award: RM1 HG009490
• NI
  National Institute of General Medical Sciences

  Award: R35 GM118062
• NI
  National Institute of Allergy and Infectious Diseases

  Award: U01 AI142756
• NI
  National Institute of Biomedical Imaging and Bioengineering

  Award: R01 EB031172