CRISPR/Cas9-mediated genome editing induces a p53-mediated DNA damage response and cell cycle arrest. Inhibiting p53 prevents this response and increases homologous recombination (HR) by more than five-fold, improving precision genome editing in normal cells. However, caution is needed in using CRISPR for human therapies until the p53 activation mechanism is understood. CRISPR/Cas9 induces DNA double-strand breaks (DSBs), which are repaired by either error-prone non-homologous-end-joining (NHEJ) or precise HR. HR is more efficient during DNA replication, while NHEJ dominates in G1 phase. In primary cells, HR is inefficient due to high NHEJ activity and apoptosis. Efforts to improve editing in primary cells include increasing repair templates, using NHEJ inhibitors, and optimizing transfection, but the underlying inefficiency remains unclear. In RPE-1 cells, CRISPR screens showed increased gRNA targeting p53, p21, and RB1, suggesting that DSBs activate p53, leading to cell cycle arrest or apoptosis. This effect was confirmed in p53-deficient cells, where HR was more efficient. Transient p53 inhibition increased HR and precision editing by allowing cell cycle progression despite DSBs. Cas9 and gRNA are constitutively active in lentiviral screens, but transient activation still triggered p53 responses. RPE-1 cells with wild-type p53 showed G1 arrest and p21 upregulation, while p53-deficient cells did not. This G1 arrest likely prevents HR, leading to imprecise NHEJ repair and reduced HR efficiency. Inhibiting p53 with MDM2 improved HR efficiency in RPE-1 cells, indicating that p53 signaling limits precision editing. However, p53 inhibition may increase the risk of chromosomal rearrangements and tumorigenic mutations. Other methods, like using adeno-associated virus (AAV) for repair DNA delivery, may also sensitize cells to DNA damage. The study shows that p53 inhibition improves HR and precision editing but may have negative consequences. Future research should focus on controlling DNA damage responses to enhance editing efficiency without promoting tumorigenic cells. Caution is advised in therapeutic CRISPR/Cas9 use due to potential risks.CRISPR/Cas9-mediated genome editing induces a p53-mediated DNA damage response and cell cycle arrest. Inhibiting p53 prevents this response and increases homologous recombination (HR) by more than five-fold, improving precision genome editing in normal cells. However, caution is needed in using CRISPR for human therapies until the p53 activation mechanism is understood. CRISPR/Cas9 induces DNA double-strand breaks (DSBs), which are repaired by either error-prone non-homologous-end-joining (NHEJ) or precise HR. HR is more efficient during DNA replication, while NHEJ dominates in G1 phase. In primary cells, HR is inefficient due to high NHEJ activity and apoptosis. Efforts to improve editing in primary cells include increasing repair templates, using NHEJ inhibitors, and optimizing transfection, but the underlying inefficiency remains unclear. In RPE-1 cells, CRISPR screens showed increased gRNA targeting p53, p21, and RB1, suggesting that DSBs activate p53, leading to cell cycle arrest or apoptosis. This effect was confirmed in p53-deficient cells, where HR was more efficient. Transient p53 inhibition increased HR and precision editing by allowing cell cycle progression despite DSBs. Cas9 and gRNA are constitutively active in lentiviral screens, but transient activation still triggered p53 responses. RPE-1 cells with wild-type p53 showed G1 arrest and p21 upregulation, while p53-deficient cells did not. This G1 arrest likely prevents HR, leading to imprecise NHEJ repair and reduced HR efficiency. Inhibiting p53 with MDM2 improved HR efficiency in RPE-1 cells, indicating that p53 signaling limits precision editing. However, p53 inhibition may increase the risk of chromosomal rearrangements and tumorigenic mutations. Other methods, like using adeno-associated virus (AAV) for repair DNA delivery, may also sensitize cells to DNA damage. The study shows that p53 inhibition improves HR and precision editing but may have negative consequences. Future research should focus on controlling DNA damage responses to enhance editing efficiency without promoting tumorigenic cells. Caution is advised in therapeutic CRISPR/Cas9 use due to potential risks.