MEGA dPCR reveals chromosomal aberrations, NHEJ precise repair, recurrent nuclease cleavage and DSB half-life
Authors: White Nathan , Chalk John , Hu Yi-Ting , Pins Samuel , Antoniou Panagiotis , Wimberger Sandra , Svensson Stina , Caetano-Silva Soraia , Mudde Anne , Raj Rajeev , Selvaraj Sridhar , Feist William , Romito Marianna , Joseph Chinnu , Sienski Grzegorz , Nitsch Roberto , Booth Claire , Santilli Giorgia , Cavazza Alessia , Porteus Matthew , Maresca Marcello , Thrasher Adrian , Turchiano Giandomenico 
Published: June 2024

Abstract

Advances in designer DNA editors, such as programmable nucleases, PASTE, Prime, and base editors, have recently proved their therapeutic value and potential to treat genetic disorders. However, a comprehensive understanding of their cellular activities is essential for improving their editing efficiency and ensuring a safe clinical translation of cell and gene therapies. A myriad of techniques have emerged to detect or anticipate genotoxic events, reflecting the increasing demand for a thorough characterisation over these editing tools’ activities. Nonetheless, these approaches have limitations, including high costs, time-consuming procedures, requirement of extensive bioinformatics knowledge, and the inference of selective and sometimes biased aberration evaluation. Moreover, the temporal dynamics of DNA editing and subsequent repairs remain poorly understood, permitting misinterpretation as well as observational biases.
To address these challenges, we introduce Multipurpose Editing and Genotoxicity Assessment (MEGA), a novel diagnostic toolkit employing multiplexed digital PCR (dPCR) assays for a comprehensive characterisation of mutations arising alongside the on- and off-target sites by gene editing tools. MEGA delivers valuable insights into genome integrity as well as quantification of episomal or integrated DNA donor templates. To demonstrate the widespread applicability of MEGA, we used numerous designer nucleases (SpCas9, TALEN, Cas12a, and PsCas9) to edit clinically relevant primary cells, including human haematopoietic stem and progenitor cells (HSPCs) pre- and post-transplantation in murine model, induced pluripotent stem cells (iPSCs), and T cells. By quantifying up to ~90% of alleles with unresolved double-strand breaks (DSBs) and other aberrations across various therapeutic gene targets, we exposed the biases of commonly used mutation-screening assays.
MEGA’s unmatched characterisation in assessing DNA status post-editing revealed many novel insights into DNA repair inhibitor effects, designer nuclease activity and DNA repair mechanisms in primary cells, including the elusive yet highly prevalent precise DSB repair via NHEJ and subsequent recurrent nuclease cleavage events. This technology also enabled us to measure the mutation probabilities of DSB resolution with unprecedented accuracy, as well as expose the kinetics associated with their resolution by DNA repair pathways.
MEGA addresses critical gaps in the available genetic engineering analysis toolkit by providing a rapid, accessible, and specific overview of genome integrity post-gene editing that can be effortlessly applied to clinically relevant samples. Furthermore, by exploiting this novel technique, we elucidated fundamental biological concepts underlying DNA repair, paving the way for extensive mechanistic studies for improving designer editor activity and gene therapy safety.