Core Viewpoint - The study highlights the unintended adverse effects of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells (HSPC), specifically focusing on senescence and inflammation, which can negatively impact the efficacy and safety of gene therapy [4][5][15]. Group 1: Gene Editing Techniques and Challenges - Gene editing in HSPC represents a transformative approach for treating immune, blood, and metabolic genetic diseases, utilizing platforms like CRISPR-Cas9 and AAV6 for targeted gene modifications [8]. - A major limitation of efficient homologous recombination (HDR) gene editing is that primitive HSCs are in a quiescent state, which hinders long-term proliferation of HDR-edited human HSPC [9][10]. - The activation of DNA damage response (DDR) due to double-strand breaks (DSB) can adversely affect the adaptability of HSPC, leading to increased sensitivity and reduced clonal diversity in xenotransplantation [10]. Group 2: Adverse Effects and Mitigation Strategies - The study found that CRISPR-Cas9/AAV6-mediated gene editing induces senescence-like phenotypes and inflammatory responses in HSPC, which impair the functionality of HDR-edited cells [12][15]. - Short-term suppression of p53 can alleviate the negative impacts of gene editing-induced DDR, but complete inactivation of p53 raises concerns about genetic toxicity [11]. - The use of Anakinra, an IL-1 signaling antagonist, has been identified as a promising strategy to enhance the clonal output of HDR-edited cells while minimizing genetic toxicity risks associated with the editing process [5][14][15]. Group 3: Research Findings and Implications - The core findings of the research indicate that CRISPR-Cas9/AAV6 gene editing leads to significant accumulation of senescence markers in HDR-edited HSPC, which can be mitigated to enhance long-term hematopoietic reconstitution [15]. - The study proposes strategies to overcome key obstacles in HDR-based gene therapy for HSPC, aiming to improve clinical efficacy and safety [6][17].