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

• Published in: Nature communications Vol. 14; no. 1; p. 4761
• Main Authors: Wimberger, Sandra, Akrap, Nina, Firth, Mike, Brengdahl, Johan, Engberg, Susanna, Schwinn, Marie K., Slater, Michael R., Lundin, Anders, Hsieh, Pei-Pei, Li, Songyuan, Cerboni, Silvia, Sumner, Jonathan, Bestas, Burcu, Schiffthaler, Bastian, Magnusson, Björn, Di Castro, Silvio, Iyer, Preeti, Bohlooly-Y, Mohammad, Machleidt, Thomas, Rees, Steve, Engkvist, Ola, Norris, Tyrell, Cadogan, Elaine B., Forment, Josep V., Šviković, Saša, Akcakaya, Pinar, Taheri-Ghahfarokhi, Amir, Maresca, Marcello
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14.08.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/106; 13/109; 45; 45/100; 45/22; 45/23; 45/41; 45/77; 49; 49/47; 631/337/1427/2190; 631/337/4041/3196; Combined treatment; CRISPR; Deoxyribonucleic acid; DNA; DNA damage; DNA polymerase; DNA repair; DNA-dependent protein kinase; DNA-directed DNA polymerase; Double-strand break repair; Editing; Efficiency; Genetic disorders; Genetic modification; Genetic screening; Genome editing; Genomes; Humanities and Social Sciences; Integration; Kinases; Libraries; Medical research; multidisciplinary; Science; Science (multidisciplinary); Target recognition
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-40344-4

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. Low efficiency of target DNA integration remains a challenge in genome engineering. Here the authors perform large-scale compound library and genetic screens to identify targets that enhance gene editing: they see that combined DNA-PK and Polϴ inhibition with potent compounds increases editing efficiency and precision.