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

Author

Listed:
  • 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
    University of Gothenburg)

  • 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
    University of Oulu)

  • 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)

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.

Suggested Citation

  • 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 & M, 2021. "Universal toxin-based selection for precise genome engineering in human cells," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-020-20810-z
    DOI: 10.1038/s41467-020-20810-z
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    Citations

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    Cited by:

    1. Sandra Wimberger & Nina Akrap & Mike Firth & Johan Brengdahl & Susanna Engberg & Marie K. Schwinn & Michael R. Slater & Anders Lundin & Pei-Pei Hsieh & Songyuan Li & Silvia Cerboni & Jonathan Sumner &, 2023. "Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    2. Marion Rosello & Malo Serafini & Luca Mignani & Dario Finazzi & Carine Giovannangeli & Marina C. Mione & Jean-Paul Concordet & Filippo Del Bene, 2022. "Disease modeling by efficient genome editing using a near PAM-less base editor in vivo," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    3. I. F. Schene & I. P. Joore & J. H. L. Baijens & R. Stevelink & G. Kok & S. Shehata & E. F. Ilcken & E. C. M. Nieuwenhuis & D. P. Bolhuis & R. C. M. Rees & S. A. Spelier & H. P. J. Doef & J. M. Beekman, 2022. "Mutation-specific reporter for optimization and enrichment of prime editing," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    4. Burcu Bestas & Sandra Wimberger & Dmitrii Degtev & Alexandra Madsen & Antje K. Rottner & Fredrik Karlsson & Sergey Naumenko & Megan Callahan & Julia Liz Touza & Margherita Francescatto & Carl Ivar Möl, 2023. "A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    5. Kasparas Petkevicius & Henrik Palmgren & Matthew S. Glover & Andrea Ahnmark & Anne-Christine Andréasson & Katja Madeyski-Bengtson & Hiroki Kawana & Erik L. Allman & Delaney Kaper & Martin Uhrbom & Lis, 2022. "TLCD1 and TLCD2 regulate cellular phosphatidylethanolamine composition and promote the progression of non-alcoholic steatohepatitis," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    6. Sébastien Levesque & Diana Mayorga & Jean-Philippe Fiset & Claudia Goupil & Alexis Duringer & Andréanne Loiselle & Eva Bouchard & Daniel Agudelo & Yannick Doyon, 2022. "Marker-free co-selection for successive rounds of prime editing in human cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    7. Martin Peterka & Nina Akrap & Songyuan Li & Sandra Wimberger & Pei-Pei Hsieh & Dmitrii Degtev & Burcu Bestas & Jack Barr & Stijn Plassche & Patricia Mendoza-Garcia & Saša Šviković & Grzegorz Sienski &, 2022. "Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing," Nature Communications, Nature, vol. 13(1), pages 1-9, December.

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