In the context of gene editing applications, genotoxicity may be defined as, “the property of an agent able to alter the genetic function within a cell causing unwanted mutations/effects, which may lead to functional impairment (e.g., cancer, therapy impairment, differentiation impairment).”1 Viewed from this perspective, there is an important advantage nickase-based editing systems like Base Editors (BEs) and Prime Editors (PEs) are understood by many to have over Cas9 nuclease-mediated systems. When BEs and PEs make changes to DNA, they are not as likely to trigger double-strand breaks (DSBs). This is important because when the cell’s DNA-repair machinery sets out to fix a DSB, there is a higher risk of genotoxic misrepair events. Despite the prevalent views described, recently published work2 scrutinizing these genetic editing tools reveals they may have a risk profile which is less favorable than previously believed, particularly in the case of BEs.

It turns out BEs especially, may have a tendency to trigger unexpected instances of DNA double-strand breaks, deletions, and translocations. Conversion of a single-strand break to a double-strand break after the transit of a DNA replication fork may be a shared mechanism among all nickase-based systems. Furthermore, the higher fraction of alleles carrying deletions and a stronger propensity to generate translocations observed for BEs likely reflect the involvement of the base excision repair (BER) pathway. These published results challenge claims of lower risk with nickase-based systems.

While PEs exhibit a lower inclination for genotoxic events compared to BEs, both editing systems elicit detrimental transcriptional responses. These responses, triggered by the genetic editing process, have cascading effects on editing efficiency and the ability of hematopoietic stem and progenitor cells (HSPCs) to repopulate. The activation of the p53 pathway, induction of pro-apoptotic transcripts, and alterations in the mutational landscape represent significant obstacles to successfully using BEs and PEs for gene editing.

The unexpected genotoxic effects highlighted in the study prompt a re-evaluation of an assumption of safety, most of all with respect to BEs. The findings also underscore the importance of continuous engineering, optimization of RNA design to mitigate interferon responses, and a comprehensive assessment of the risk-benefit ratio.

While BEs and PEs represent powerful tools for genetic engineering, the newfound concerns regarding unintended DNA changes and genotoxic effects warrant caution and further study. The mechanisms underlying genotoxic impacts, as well as the frequency and extent of those impacts under different conditions must be understood. By alerting the scientific community of possible overlooked risks, the published work may cause those employing these tools to devise strategies to minimize the unintended effects described, and to control for them in experiments. This will be to the benefit of all, as their important work is aided by these new insights.



  1. Blattner, G., Cavazza, A., Thrasher, A. J., & Turchiano, G. (2020). Gene Editing and Genotoxicity: Targeting the Off-Targets. Frontiers in genome editing, 2, 613252.
  2. Fiumara, M., Ferrari, S., Omer-Javed, A., Beretta, S., Albano, L., Canarutto, D., … & Naldini, L. (2023). Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nature biotechnology, 1-15.