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

Publication Date: 14 August 2023

Wimberger, S. et al. (2023) Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing. Nat Commun. 14, 4761. DOI: 10.1038/s41467-023-40344-4

In recent years, genome editing with CRISPR/Cas9 technology has been a valuable tool for biomedical research. Pre-clinical and clinical studies have highlighted the potential use of CRISPR/Cas9 to treat genetics disorders. The most common strategy for genome editing uses single-guide RNA (sgRNA) directed DNA double-strand breaks (DSB). The DSB then triggers DSB repair pathways that each have different gene editing outcomes. A major challenge facing precise gene editing is that the DNA repair pathway yielding the most precise editing, homology directed repair (HDR), is outperformed by other repair pathways that form insertions and deletions (InDels) and imprecise edits, such as nonhomologous end joining (NHEJ) and alternative end joining (alt-EJ).

Previous studies have identified that NHEJ is the quickest and most common DSB repair pathway. It requires the DNA-dependent protein kinase catalytic subunits (DNA-PKcs) to form the DNA-PK complex that activates other NHEJ factors. Alt-EJ utilizes a process called end resection which shares initial steps with HDR. In most eukaryotic cells, DNA polymerase theta (Polϴ) is the main mediator of alt-EJ. DNA-PK inhibitors have been found to decrease NHEJ and enhance HDR. Additionally, knockdown and knockout of Polϴ has been shown to reduce translocations and large deletions. Though previous research has successfully used NHEJ and alt-EJ for precise end joining repair, the flexibility of HDR makes it an attractive strategy for precise genome editing.

This study used high-throughput screening to identify compounds that enhance HDR and reduce side effects associated with Cas9. Researchers identified AZD7648 as the most potent and selective DNA-PK inhibitor. AZD7648 is part of a newer generation of DNA-PK inhibitors and is characterized by higher selectivity and improved knock-in efficiency. Additionally, they found that AZD7648 increases ssDNA integration and reduces NHEJ-mediated deletions. To further increase bias towards HDR, researchers tested Polϴ in combination with AZD7648 for increased integration efficiency. Results of this study demonstrated that combined treatment of Polϴ inhibitors and AZD7648 (2iHDR) improve the performance and precision of knock-in gene editing. Further research is required to fully understand potential undesirable side effects of this strategy.

Keywords: CRISPR/Cas9, nonhomologous end joining (NHEJ), homology directed repair (HDR), alternative end joining (alt-EJ), gene editing, genome editing