College of Pharmacy, Seoul National University, Seoul, Republic of Korea.
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.
Nat Commun. 2024 May 11;15(1):4002. doi: 10.1038/s41467-024-48111-9.
Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
精确的基因组编辑对于建立同基因的人类疾病模型和从患者来源的 hPSCs 进行体外干细胞治疗至关重要。与 Cas9 介导的基因敲入不同，胞嘧啶碱基编辑器和 Prime 编辑器在不诱导 DNA 双链断裂的情况下实现了理想的基因校正。然而，hPSCs 具有高度活跃的 DNA 修复途径，特别容易受到 p53 依赖性细胞死亡的影响。这些独特的特征阻碍了基因编辑在 hPSCs 中的效率。在这里，我们证明了 p53 介导的细胞死亡的双重抑制和 DNA 损伤修复系统的独特激活，在胞嘧啶碱基编辑器或 Prime 编辑器引起的 DNA 损伤后，可累加性地提高 hPSCs 中的编辑效率。BE4stem 系统由 p53DD（一种显性失活的 p53）和三种 UNG 抑制剂组成，专门设计用于减少碱基切除修复，可提高 hPSCs 中胞嘧啶碱基编辑器的效率。在传统的 Prime 编辑器系统中添加显性失活的 MLH1 以抑制错配修复活性和 p53DD，也可显著提高 hPSCs 中 Prime 编辑器的效率。因此，在基因编辑过程中结合抑制 hPSCs 中涉及的不同细胞级联反应，可以显著提高这些细胞中精确的基因组编辑效率。
