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

Songyuan Li (), Nina Akrap, Silvia Cerboni, Michelle J. Porritt, Sandra Wimberger, Anders Lundin, Carl Möller, Mike Firth, Euan Gordon, Bojana Lazovic, Aleksandra Sieńska, Luna Simona Pane, Matthew A. Coelho, Giovanni Ciotta, Giovanni Pellegrini, Marcella Sini, Xiufeng Xu, Suman Mitra, Mohammad Bohlooly-Y, Benjamin J. M. Taylor, Grzegorz Sienski () and Marcello Maresca ()
Additional contact information
Songyuan Li: BioPharmaceuticals R&D, AstraZeneca
Nina Akrap: BioPharmaceuticals R&D, AstraZeneca
Silvia Cerboni: Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca
Michelle J. Porritt: BioPharmaceuticals R&D, AstraZeneca
Sandra Wimberger: BioPharmaceuticals R&D, AstraZeneca
Anders Lundin: BioPharmaceuticals R&D, AstraZeneca
Carl Möller: BioPharmaceuticals R&D, AstraZeneca
Mike Firth: R&D Data Infrastructure & Tools, AstraZeneca
Euan Gordon: BioPharmaceuticals R&D, AstraZeneca
Bojana Lazovic: BioPharmaceuticals R&D, AstraZeneca
Aleksandra Sieńska: BioPharmaceuticals R&D, AstraZeneca
Luna Simona Pane: BioPharmaceuticals R&D, AstraZeneca
Matthew A. Coelho: Wellcome Sanger Institute
Giovanni Ciotta: BioPharmaceuticals R&D, AstraZeneca
Giovanni Pellegrini: BioPharmaceuticals R&D, AstraZeneca
Marcella Sini: BioPharmaceuticals R&D, AstraZeneca
Xiufeng Xu: Karolinska Institute
Suman Mitra: Inserm UMR1277 CNRS UMR9020 – CANTHER, Institut pour la Recherche sur le Cancer de Lille
Mohammad Bohlooly-Y: BioPharmaceuticals R&D, AstraZeneca
Benjamin J. M. Taylor: BioPharmaceuticals R&D, AstraZeneca
Grzegorz Sienski: BioPharmaceuticals R&D, AstraZeneca
Marcello Maresca: BioPharmaceuticals R&D, AstraZeneca

Nature Communications, 2021, vol. 12, issue 1, 1-14

Abstract: Abstract 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.

Date: 2021
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DOI: 10.1038/s41467-020-20810-z

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