2. Academic Validation
  3. Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing

Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing

  • Nat Commun. 2023 Aug 14;14(1):4761. doi: 10.1038/s41467-023-40344-4.
Sandra Wimberger 1 2,  Nina Akrap 3,  Mike Firth 4,  Johan Brengdahl 5,  Susanna Engberg 6,  Marie K Schwinn 7,  Michael R Slater 7,  Anders Lundin 8,  Pei-Pei Hsieh 3,  Songyuan Li 3,  Silvia Cerboni 9,  Jonathan Sumner 10,  Burcu Bestas 3,  Bastian Schiffthaler 11,  Björn Magnusson 8,  Silvio Di Castro 12,  Preeti Iyer 13,  Mohammad Bohlooly-Y 8,  Thomas Machleidt 7,  Steve Rees 14,  Ola Engkvist 13,  Tyrell Norris 6,  Elaine B Cadogan 15,  Josep V Forment 15,  Saša Šviković 3,  Pinar Akcakaya 3,  Amir Taheri-Ghahfarokhi 3,  Marcello Maresca 16
Affiliations

Affiliations

  • 1 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. sandra.wimberger@astrazeneca.com.
  • 2 Department of Chemistry & Molecular Biology, University of Gothenburg, Gothenburg, Sweden. sandra.wimberger@astrazeneca.com.
  • 3 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 4 Data Sciences & Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
  • 5 Cell Assay Development, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 6 Cell Engineering Sweden, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 7 Promega Corporation, Madison, WI, USA.
  • 8 Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 9 Translational Science & Experimental Medicine, Research and Early Development, Respiratory & Immunology (R&I), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 10 Cell Immunology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
  • 11 Data Sciences & Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 12 Compound Synthesis & Management, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 13 Molecular AI, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 14 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
  • 15 Bioscience, Early Oncology, AstraZeneca, Cambridge, UK.
  • 16 Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. marcello.maresca@astrazeneca.com.
PMID: 37580318 DOI: 10.1038/s41467-023-40344-4
Abstract

Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK Inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA Polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.

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