Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells
Abstract Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integ...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-08-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-63164-0 |
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| author | George D. Lampe Ashley R. Liang Dennis J. Zhang Israel S. Fernández Samuel H. Sternberg |
| author_facet | George D. Lampe Ashley R. Liang Dennis J. Zhang Israel S. Fernández Samuel H. Sternberg |
| author_sort | George D. Lampe |
| collection | DOAJ |
| description | Abstract Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications. |
| format | Article |
| id | doaj-art-354eb574abb24762b435684bf3e5cfae |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-354eb574abb24762b435684bf3e5cfae2025-08-24T11:36:44ZengNature PortfolioNature Communications2041-17232025-08-0116111410.1038/s41467-025-63164-0Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cellsGeorge D. Lampe0Ashley R. Liang1Dennis J. Zhang2Israel S. Fernández3Samuel H. Sternberg4Department of Biochemistry and Molecular Biophysics, Columbia UniversityDepartment of Biochemistry and Molecular Biophysics, Columbia UniversityDepartment of Biochemistry and Molecular Biophysics, Columbia UniversityIkerbasque, Basque Foundation for ScienceDepartment of Biochemistry and Molecular Biophysics, Columbia UniversityAbstract Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications.https://doi.org/10.1038/s41467-025-63164-0 |
| spellingShingle | George D. Lampe Ashley R. Liang Dennis J. Zhang Israel S. Fernández Samuel H. Sternberg Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells Nature Communications |
| title | Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells |
| title_full | Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells |
| title_fullStr | Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells |
| title_full_unstemmed | Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells |
| title_short | Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells |
| title_sort | structure guided engineering of type i f casts for targeted gene insertion in human cells |
| url | https://doi.org/10.1038/s41467-025-63164-0 |
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