Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase

• 成果类型: Article
• 署名作者: Witte, Isaac P.; Lampe, George D.; Eitzinger, Simon; Miller, Shannon M.; Berrios, Kiara N.; McElroy, Amber N.; King, Rebeca T.; Stringham, Olivia G.; Gelsinger, Diego R.; Vo, Phuc Leo H.; Chen, Albert T.; Tolar, Jakub; Osborn, Mark J.; Sternberg, Samuel H.; Liu, David R.
• 署名单位: Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University; Howard Hughes Medical Institute; Harvard University; Columbia University; Howard Hughes Medical Institute; Columbia University; University of Minnesota System; University of Minnesota Twin Cities; Scripps Research Institute; Vertex Pharmaceuticals
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-8092
• DOI: 10.1126/science.adt5199
• 发表日期: 2025-05-15
• 关键词: continuous directed evolution; phage-assisted evolution; dna-binding proteins; human genome; base; therapy; stringency; expression; selection; efficacy

摘要

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 similar to 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.