Gene editing in human pluripotent stem cells (hPSC) is a powerful tool for understanding biology, for drug discovery and gene therapy. Naïve hPSC have been suggested to be superior for gene editing compared to conventional ‘primed’ hPSC. Using droplet digital PCR, we uncover the kinetics of Cas9-induced double strand break repair in conventional hPSC. Cut but unrepaired alleles reach their maximum after 12–24 h. Homology directed repair plateaus after 24 h, whereas repair by non-homologous end joining continues until 48 h after Cas9 introduction. Using this method, we demonstrate that the rate of homology directed repair to resolve Cas9-induced double strand breaks is 40% lower in naïve hPSC compared to conventional hPSC, correlating with, and feasibly explained by, a higher number of cells in G₁ phase of the cell cycle in naïve hPSC. Therefore, naïve hPSC are less efficient for CRISPR/Cas9-mediated homology directed repair.

人类多能干细胞（hPSC）中的基因编辑是了解生物学，进行药物发现和基因治疗的强大工具。与传统的“初免” hPSC相比，朴素的hPSC已被认为在基因编辑方面更胜一筹。使用液滴数字PCR，我们发现了常规hPSC中Cas9诱导的双链断裂修复的动力学。剪切但未修复的等位基因在12-24小时后达到最大值。同源性指导修复平台在24小时后达到稳定状态，而通过非同源末端连接的修复持续到引入Cas9后48小时。使用这种方法，我们证明与传统的hPSC相比，纯朴的hPSC中针对Cas9诱导的双链断裂的同源性定向修复率要低40％，这与G _1中的细胞数量更多有关，并可以用其解释幼稚hPSC中细胞周期的阶段。因此，单纯hPSC在CRISPR / Cas9介导的同源性定向修复中效率较低。