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Universal toxin-based selection for precise genome engineering in human cells
- Li, S. Y. (author)
- Akrap, N. (author)
- Cerboni, S. (author)
- show more...
- Porritt, M. J. (author)
- Lundin, A. (author)
- Moller, C. (author)
- Firth, M. (author)
- Gordon, E. (author)
- Lazovic, B. (author)
- Sienska, A. (author)
- Pane, L. S. (author)
- Coelho, M. A. (author)
- Ciotta, G. (author)
- Pellegrini, G. (author)
- Sini, M. (author)
- Xu, X. F. (author)
- Mitra, S. (author)
- Bohlooly-Y, M. (author)
- Taylor, B. J. M. (author)
- Sienski, G. (author)
- Maresca, M. (author)
- show less...
- (creator_code:org_t)
- 2021-01-21
- 2021
- English.
- In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
- Journal article (peer-reviewed)
Abstract
Subject headings
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- Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems. Genome engineering in cell lines or human stem cells often has poor efficiency, limiting the development of research and therapeutic applications. Here, the authors use a toxin-based selection system for precise bi-allelic engineering in cells and in vivo.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Keyword
- epidermal-growth-factor
- diphtheria-toxin
- heparin-binding
- targeted
- integration
- transient reporter
- stem-cells
- receptor
- DNA
- immunotherapy
- mechanisms
- Science & Technology - Other Topics
Publication and Content Type
- ref (subject category)
- art (subject category)
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- Li, S. Y.
- Akrap, N.
- Cerboni, S.
- Porritt, M. J.
- Wimberger, Sandr ...
- Lundin, A.
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- Moller, C.
- Firth, M.
- Gordon, E.
- Lazovic, B.
- Sienska, A.
- Pane, L. S.
- Coelho, M. A.
- Ciotta, G.
- Pellegrini, G.
- Sini, M.
- Xu, X. F.
- Mitra, S.
- Bohlooly-Y, M.
- Taylor, B. J. M.
- Sienski, G.
- Maresca, M.
- show less...
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- University of Gothenburg
- Karolinska Institutet
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