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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.

Original publication




Journal article


Stem Cell Res

Publication Date





Gene editing efficiency, Ground state, HDR, HR, Homologous recombination, Homology directed repair, Human, NHEJ, Naïve, Non-homologous end joining, Pluripotency, Pluripotent, Stem cell