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. Overall design: 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.

体外激活是基于造血干祖细胞（hematopoietic stem and progenitor cell, HSPC）的临床应用中，通过同源定向修复（homology-driven repair, HDR）实现足量基因编辑的必要前提。本研究发现，缩短培养时间可缓解p53介导的、针对CRISPR/Cas9诱导DNA双链断裂的DNA损伤应答，进而提升编辑后造血干祖细胞的造血重建能力。但该操作会导致同源定向修复效率降低，使得体外培养虽为必需却又存在不利影响。从机制层面来看，体外激活会触发由p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38 MAPK）磷酸化启动的多步骤过程：该过程会生成有丝分裂活性的活性氧（reactive oxygen species, ROS），促进细胞周期快速进程及后续增殖相关的DNA损伤。因此，在基因编辑前抑制p38，可延迟细胞周期G1/S期转换，并扩增转录层面定义的造血干细胞（hematopoietic stem cell, HSC）群体，最终使编辑后的细胞具备更优异的多系分化能力、连续移植后的持续留存能力、更丰富的多克隆谱系，以及保存更完好的基因组完整性。本研究数据证实，增殖应激是造血干祖细胞功能异常的驱动因素，这为设计更高效、更安全的临床基因校正策略提供了关键理论依据。实验整体设计：针对各造血干祖细胞亚群，对未编辑、仅转染Cas9的造血干祖细胞、以及经腺相关病毒6型（adeno-associated virus 6, AAV6）介导（GFP-/GFP+）编辑的细胞，施加p38抑制剂（p38 inhibitor, p38i）处理后，进行液滴法单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。为提升更原始造血干细胞的检出率，我们分选了富集造血干细胞的细胞群（定义为CD34+CD133+CD90+CD45RA-），并在进行hashtag标记后将其与整体样本群体混合。