Transient ATM inhibition enhances knock-in efficiency in hematopoietic stem cells by attenuating the DNA damage response

Abstract

Precise genome editing in hematopoietic stem cells (HSCs) offers great potential for treating inherited blood disorders, but low knock-in (KI) efficiency, due to HSC quiescence and a preference for non-homologous end joining (NHEJ) and DNA damage-induced apoptosis, remains a major barrier. Here, we demonstrate that transient inhibition of Ataxia-Telangiectasia Mutated (ATM) kinase markedly enhances KI efficiency in mouse HSCs genome-edited with Cas9/RNP and AAV donor DNA. Phosphoproteomic analysis and capillary western blotting revealed that ATM inhibition suppressed the Cas9-AAV-induced ATM activation and subsequent DNA damage response, reduced p53-dependent apoptosis and preserved knock-in competent cells. In transplantation experiments, ATM inhibition preserved long-term engrafting genome-edited HSCs, increasing their frequency from [~]0.3% to [~]40% in secondary recipients - a >100-fold enhancement compared to untreated cells. Furthermore, in an X-SCID mouse model, ATM inhibition enhanced KI efficiency and restored expression of IL-2 receptor {gamma} chain (CD132). These strikingly novel findings highlight transient ATM inhibition as a powerful and clinically relevant approach to enhance KI-mediated genome editing in HSCs, while preserving their long-term repopulating capacity. Key pointsO_LIATM inhibition enhances knock-in efficiency in mouse hematopoietic stem cells C_LIO_LIATM inhibition suppresses Cas9-AAV-induced overactivation of ATM and subsequent p53-dependent apoptosis. C_LI