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

Isaac P Witte; George D Lampe; Simon Eitzinger; Shannon M Miller; Kiara N Berríos; Amber N McElroy; Rebeca T King; Olivia G Stringham; Diego R Gelsinger; Phuc Leo H Vo; Albert T Chen; Jakub Tolar; Mark J Osborn; Samuel H Sternberg; David R Liu

doi:10.1126/science.adt5199

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

Science. 2025 May 15;388(6748):eadt5199. doi: 10.1126/science.adt5199. Epub 2025 May 15.

Authors

Isaac P Witte^#^{1

2

3}, George D Lampe^#^{4

5}, Simon Eitzinger^#^{1

2

3}, Shannon M Miller^{1

2

3}, Kiara N Berríos^{1

2

3}, Amber N McElroy⁶, Rebeca T King⁴, Olivia G Stringham^{1

2

3}, Diego R Gelsinger⁴, Phuc Leo H Vo⁴, Albert T Chen^{1

2

3}, Jakub Tolar⁶, Mark J Osborn⁶, Samuel H Sternberg^{4

5}, David R Liu^{1

2

3}

Affiliations

¹ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
³ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁴ Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
⁵ Howard Hughes Medical Institute, Columbia University, New York, NY, USA.
⁶ Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.

^# Contributed equally.

PMID: 40373119
DOI: 10.1126/science.adt5199

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.

MeSH terms

CRISPR-Cas Systems*
Directed Molecular Evolution* / methods
Gene Editing* / methods
HEK293 Cells
Humans
Mutagenesis, Insertional*
RNA, Guide, CRISPR-Cas Systems
Transposases* / genetics
Transposases* / metabolism

Substances

Transposases
RNA, Guide, CRISPR-Cas Systems