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

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, Burcu Bestas, Bastian Schiffthaler, Björn Magnusson, Silvio Di Castro, Preeti Iyer, Mohammad Bohlooly-Y, Thomas Machleidt, Steve Rees, Ola Engkvist, Tyrell Norris, Elaine B. Cadogan, Josep V. Forment, Saša Šviković, Pinar Akcakaya, Amir Taheri-Ghahfarokhi and Marcello Maresca ()
Additional contact information
Sandra Wimberger: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Nina Akrap: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Mike Firth: Data Sciences & Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Johan Brengdahl: Cell Assay Development, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Susanna Engberg: Cell Engineering Sweden, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Marie K. Schwinn: Promega Corporation
Michael R. Slater: Promega Corporation
Anders Lundin: Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Pei-Pei Hsieh: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Songyuan Li: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Silvia Cerboni: Translational Science & Experimental Medicine, Research and Early Development, Respiratory & Immunology (R&I), BioPharmaceuticals R&D, AstraZeneca
Jonathan Sumner: Cell Immunology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Burcu Bestas: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Bastian Schiffthaler: Data Sciences & Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Björn Magnusson: Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Silvio Di Castro: Compound Synthesis & Management, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Preeti Iyer: Molecular AI, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Mohammad Bohlooly-Y: Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Thomas Machleidt: Promega Corporation
Steve Rees: Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Ola Engkvist: Molecular AI, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Tyrell Norris: Cell Engineering Sweden, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Elaine B. Cadogan: Bioscience, Early Oncology, AstraZeneca
Josep V. Forment: Bioscience, Early Oncology, AstraZeneca
Saša Šviković: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Pinar Akcakaya: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Amir Taheri-Ghahfarokhi: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca
Marcello Maresca: Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca

Nature Communications, 2023, vol. 14, issue 1, 1-18

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-40344-4

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