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

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

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

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.

Original language	English (US)
Article number	eadt5199
Journal	Science
Volume	388
Issue number	6748
DOIs	https://doi.org/10.1126/science.adt5199
State	Published - May 15 2025

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Witte, I. P., Lampe, G. D., Eitzinger, S., Miller, S. M., Berríos, K. N., McElroy, A. N., King, R. T., Stringham, O. G., Gelsinger, D. R., Vo, P. L. H., Chen, A. T., Tolar, J., Osborn, M. J., Sternberg, S. H., & Liu, D. R. (2025). Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science, 388(6748), Article eadt5199. https://doi.org/10.1126/science.adt5199

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title = "Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase",

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 \textasciitilde{}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.",

author = "Witte, \{Isaac P.\} and Lampe, \{George D.\} and Simon Eitzinger and Miller, \{Shannon M.\} and Berr{\'i}os, \{Kiara N.\} and McElroy, \{Amber N.\} and King, \{Rebeca T.\} and Stringham, \{Olivia G.\} and Gelsinger, \{Diego R.\} and Vo, \{Phuc Leo H.\} and Chen, \{Albert T.\} and Jakub Tolar and Osborn, \{Mark J.\} and Sternberg, \{Samuel H.\} and Liu, \{David R.\}",

note = "Publisher Copyright: Copyright {\textcopyright} 2025 the authors, some rights reserved.",

year = "2025",

month = may,

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doi = "10.1126/science.adt5199",

language = "English (US)",

volume = "388",

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AU - Witte, Isaac P.

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AU - Miller, Shannon M.

AU - Berríos, Kiara N.

AU - McElroy, Amber N.

AU - King, Rebeca T.

AU - Stringham, Olivia G.

AU - Gelsinger, Diego R.

AU - Vo, Phuc Leo H.

AU - Chen, Albert T.

AU - Tolar, Jakub

AU - Osborn, Mark J.

AU - Sternberg, Samuel H.

AU - Liu, David R.

PY - 2025/5/15

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N2 - 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.

AB - 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.

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

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