University of Oulu

Li, S., Akrap, N., Cerboni, S. et al. Universal toxin-based selection for precise genome engineering in human cells. Nat Commun 12, 497 (2021).

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

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Author: Li, Songyuan1; Akrap, Nina1; Cerboni, Silvia2;
Organizations: 1Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
2Translational Science and Experimental Medicine, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
3Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden
4R&D Data Infrastructure & Tools, AstraZeneca, Cambridge, UK
5Discovery Biology SWE, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
6Oulu Center for Cell‐Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
7Wellcome Sanger Institute, Cambridge, UK
8Discovery Biology UK, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
9CVRM pathology, Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
10Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden
11Inserm UMR1277 CNRS UMR9020 – CANTHER, Institut pour la Recherche sur le Cancer de Lille, Lille, France
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 1.7 MB)
Persistent link:
Language: English
Published: Springer Nature, 2021
Publish Date: 2023-06-28


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.

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Series: Nature communications
ISSN: 2041-1723
ISSN-E: 2041-1723
ISSN-L: 2041-1723
Volume: 12
Issue: 1
Article number: 497
DOI: 10.1038/s41467-020-20810-z
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 765269 (S.W) and 814316 (B.L.).
EU Grant Number: (814316) V.A. Cure - A multidisciplinary approach towards sustainable improvement in rare diseases care uniting Europe’s top class vascular research to find new treatment strategies for vascular anomalies
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