Enhancers of CRISPR-mediated HDR: is it worth the risk?

AZD7648 is a potent inhibitor of DNA-dependent protein kinases (DNA-PKs) and has been shown to lead to tumor regression when combined with radiotherapy.

Interestingly, AZD7648 also enhances CRISPR-mediated HDR efficiency in cultured cells and animal models by inhibiting DNA-PK (which promotes non-homologous end joining (NHEJ) and suppresses HDR). While HDR enhancement has potential clinical applications, the risks of off-target effects of CRISPR-mediated HDR using AZD7648 have not been explored so far.

The lab of Jacob Corn at ETH Zurich has now published a study which explores the genome-wide effects of AZD7648 on CRISPR–Cas9-directed genome editing.

1. Increased HDR but with Risks: The use of AZD7648 significantly improved HDR efficiencies in multiple cell types and across various loci, as measured by short-read sequencing. However, this increase in HDR was accompanied by large-scale genomic instability, manifesting as kilobase-scale deletions, chromosome arm loss, and translocations. These alterations were not detected by typical short-read sequencing methods, highlighting the limitations of this approach for evaluating genome editing outcomes.
2. Large Deletions and Chromosomal Aberrations: Long-read sequencing and other advanced methods revealed that AZD7648 treatment induced frequent kilobase-scale deletions and even megabase-scale chromosomal changes. These large-scale alterations were detected in both transformed and primary human cells.
3. Translocations and Genomic Instability: Editing with AZD7648 not only increased HDR but also the occurrence of translocations. This was particularly evident when multiple loci were targeted simultaneously, but the study also found that single-target editing with AZD7648 could induce translocations, as revealed by CAST-seq.
4. Impact on Larger Genomic Regions: In addition to small-scale deletions, AZD7648 caused the loss of large genomic regions. In upper airway organoids and HSPCs, up to 47.8% of cells exhibited gene expression loss in regions spanning several megabases, indicative of large chromosomal deletions.
5. Role of MMEJ and PolQi2 Inhibition: The study identified that the large deletions induced by AZD7648 might be mediated through microhomology-mediated end joining (MMEJ), a DNA repair pathway. Inhibiting this pathway with PolQi2, a potent inhibitor of the MMEJ helicase activity, partially reduced the kilobase-scale deletions, although it did not mitigate the megabase-scale deletions caused by AZD7648.

The results underscore the potential risks of using AZD7648 in genome editing, especially in clinical settings. The increased HDR efficiency comes at the cost of inducing large-scale genomic instability that is not readily detectable using conventional short-read sequencing. This has important implications for the safety of gene-editing therapies, as such unintended genomic alterations may not be immediately apparent but could have significant consequences for cell function and stability.

The study advocates for a more comprehensive approach to monitoring genome editing outcomes, urging the use of long-read sequencing, translocation detection, and single-cell RNA sequencing to identify large-scale genetic changes that might otherwise be overlooked. Moreover, while combining AZD7648 with inhibitors of MMEJ like PolQi2 may help reduce some of the genomic instability, the risk of megabase-scale deletions remains, which needs to be addressed before applying these methods in clinical settings.

Cullot, G., Aird, E.J., Schlapansky, M.F. et al. Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02488-6