Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 40373119.
- Also identified by DOI 10.1126/science.adt5199 and PMC identifier 12326709.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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.
Medical subject headings
- Transposases
- CRISPR-Cas Systems
- Directed Molecular Evolution
- Gene Editing
- Mutagenesis, Insertional