Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase
Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.
Authors
- Phuc Leo H. Vo (ORCID: https://orcid.org/0000-0002-3719-7770)
- Rebeca T. King (ORCID: https://orcid.org/0000-0001-5712-4539)
- Albert T. Chen (ORCID: https://orcid.org/0000-0002-5387-0208)
- Mark J. Osborn (ORCID: https://orcid.org/0000-0003-1790-0841)
- Shannon M. Miller
- George D. Lampe (ORCID: https://orcid.org/0000-0002-2873-2372)
- Simon Eitzinger (ORCID: https://orcid.org/0000-0002-2215-4348)
- Isaac P. Witte (ORCID: https://orcid.org/0000-0002-3879-0306)
- Kiara N. Berríos (ORCID: https://orcid.org/0000-0002-0684-5413)
- Jakub Tolar (ORCID: https://orcid.org/0000-0002-0957-4380)
- David R. Liu (ORCID: https://orcid.org/0000-0002-9943-7557)
- Samuel H. Sternberg (ORCID: https://orcid.org/0000-0001-8240-9114)
- Amber McElroy (ORCID: https://orcid.org/0000-0002-4169-0015)
- Diego R. Gelsinger (ORCID: https://orcid.org/0000-0002-3786-1205)
- Olivia G. Stringham
Institutions
- Broad Institute (US)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- University of Minnesota Medical Center (US)
- Columbia University (US)
Publication Details
- Journal
- Science
- Published
- 2025-05-15
- DOI
- https://doi.org/10.1126/science.adt5199
- Citations
- 52
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 41.91