# ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways

## TL;DR

Inhibiting ATM improves gene insertion efficiency in genome editing by reducing cell death and unwanted DNA repair pathways.

## Contribution

ATM inhibition enhances knock-in efficiency with AAV donors by suppressing apoptosis and classical non-homologous end-joining.

## Key findings

- ATM inhibition with AZD1390 increases knock-in efficiency with linear AAV donors.
- ATM inhibition suppresses the ATM-p53-caspase 3 apoptotic pathway in cells with high donor DNA.
- Donor DNA configuration critically influences DNA damage response signaling.

## Abstract

CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.

Linear donor DNA, including AAV, overactivates ATM in a copy-number-dependent manner, causing apoptosis and shifting repair toward c-NHEJ. ATM inhibition rescues high-donor cells and suppresses c-NHEJ, thereby increasing knock-in efficiency.