A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR/Cas9 gene editing in Hematopoietic Stem and Progenitor Cells

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-driven repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR/Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 MAPK phosphorylation, which generates mitogenic ROS, promoting fast cell cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally-defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. Droplet-based single-cell RNA sequencing (scRNA-seq) of non edited, HS Cas9, HS+AAV6(GFP-/GFP+) edited cells treated with p38i treatment across HSPC subsets. To enhance the representation of the more primitive HSCs, we sorted a pool of HSC-enriched cells (defined as CD34+CD133+CD90+CD45RA-) and mixed them with the bulk sample population upon hashtag marking.