Universal toxin-based selection for precise genome engineering in human cells.

Autor:	Li S; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. songyuan.li@astrazeneca.com., Akrap N; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Cerboni S; Translational Science and Experimental Medicine, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Porritt MJ; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Wimberger S; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.; Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden., Lundin A; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Möller C; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Firth M; R&D Data Infrastructure & Tools, AstraZeneca, Cambridge, UK., Gordon E; Discovery Biology SWE, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Lazovic B; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.; Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland., Sieńska A; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Pane LS; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Coelho MA; Wellcome Sanger Institute, Cambridge, UK., Ciotta G; Discovery Biology UK, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK., Pellegrini G; CVRM pathology, Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Sini M; CVRM pathology, Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Xu X; Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden., Mitra S; Inserm UMR1277 CNRS UMR9020 - CANTHER, Institut pour la Recherche sur le Cancer de Lille, Lille, France., Bohlooly-Y M; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden., Taylor BJM; Discovery Biology UK, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK., Sienski G; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. grzegorz.sienski@astrazeneca.com., Maresca M; Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. Marcello.Maresca@astrazeneca.com.
Jazyk:	angličtina
Zdroj:	Nature communications [Nat Commun] 2021 Jan 21; Vol. 12 (1), pp. 497. Date of Electronic Publication: 2021 Jan 21.
DOI:	10.1038/s41467-020-20810-z
Abstrakt:	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.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu