C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)
191: Efficient and Versatile Programmable Large-Gene Integration by Evolved Recombinases and Prime Editing
Type: Oral Abstract Session
Presentation Details
Session Title: Targeted Gene Insertion
Current methods to achieve targeted integration of multi-kilobase DNA sequences into mammalian genomes suffer from low efficiencies, specificities, or programmability, limiting their usage in synthetic biology, biotechnology, and gene therapies. To overcome these challenges, we developed a phage-assisted continuous evolution (PACE) selection system for recombinases, then applied the system to substantially improve the activity of large serine recombinase (LSR) for prime editing-assisted site-specific integrase gene editing (PASSIGE), an approach that couples the programmability of prime editing with the large-gene cargo compatibility of recombinases to precisely integrate large DNA cargoes into the mammalian genome. PACE-evolved LSR variants mediate up to 60% donor integration in human cell lines with pre-installed recombinase landing sites. When used in single-transfection experiments with prime editing, the evolved recombinase variants can consistently achieve targeted gene integration efficiencies of ≥30% (up to 50% without any selection or cell sorting) at therapeutically relevant sites in human cells. PASSIGE with the resulting evolved and engineered recombinase variant (eePASSIGE) substantially outperforms previously reported methods for programmable large gene integration—outperforming PASTE and the original PASSIGE system by an average of 16-fold and 4.2-fold, respectively across 12 genomic loci, including eight site of therapeutic relevance, across three human and mouse cell types. To our knowledge, the evolved and engineered Bxb1 variants generated in this study enable the most effective targeted gene integration in mammalian cells reported to date. We anticipate that these recombinase variants may also be applied to modify DNA for other applications in biology and therapeutic development. Finally, the recombinase continuous evolution system developed in this study can also be used to rapidly improve the activity of other recently discovered integrases to enhance their activity. X.D.G. and S.P. contributed equally to this work.
Xin D. Gao1,2,3, Smriti Pandey1,2,3, Nicholas A. Krasnow1,2,3, Amber McElroy4, Jordyn E. Duby1,2,3, Sarah Pierce1,2,3, Jakub Tolar4, Mark J. Osborn4, David R. Liu1,2,3
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA,2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA,3Howard Hughes Medical Institute, Harvard University, Cambridge, MA,4Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN"
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