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-agnostictreatments for loss-of-function genetic diseases and facilitate many applications in the lifesciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration butthus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution(PACE), we identified CAST variants with over 200-fold average improved integration activity. Theevolved CAST system (evoCAST) achieves ~10-30% integration efficiencies of kilobase-sizeDNA 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 undetectedindels and low levels of off-target integration. Collectively, our findings establish a platform forthe laboratory evolution of CASTs and advance a versatile system for programmable geneintegration in living systems.