A study by Steven Lin and colleagues presents a method to enhance homology-directed repair (HDR) in human cells using controlled timing of CRISPR/Cas9 delivery. By synchronizing cells in the cell cycle and delivering Cas9-guide RNA ribonucleoprotein (RNP) complexes at the optimal phase for HDR, they achieved significantly higher HDR efficiency. This approach increased HDR rates in HEK293T cells up to 38%, with high fidelity and minimal off-target effects. The method was also effective in human primary neonatal fibroblasts and human embryonic stem cells, demonstrating its broad applicability. The study shows that timing of Cas9 delivery to the G2 phase, when sister chromatids are available, enhances HDR efficiency. This strategy improves the precision and efficiency of genome editing in human cells, enabling targeted mutations, repairs, and tagging of endogenous loci. The findings highlight the importance of cell cycle synchronization in optimizing HDR and reducing off-target effects, providing a robust and effective method for human genome engineering.A study by Steven Lin and colleagues presents a method to enhance homology-directed repair (HDR) in human cells using controlled timing of CRISPR/Cas9 delivery. By synchronizing cells in the cell cycle and delivering Cas9-guide RNA ribonucleoprotein (RNP) complexes at the optimal phase for HDR, they achieved significantly higher HDR efficiency. This approach increased HDR rates in HEK293T cells up to 38%, with high fidelity and minimal off-target effects. The method was also effective in human primary neonatal fibroblasts and human embryonic stem cells, demonstrating its broad applicability. The study shows that timing of Cas9 delivery to the G2 phase, when sister chromatids are available, enhances HDR efficiency. This strategy improves the precision and efficiency of genome editing in human cells, enabling targeted mutations, repairs, and tagging of endogenous loci. The findings highlight the importance of cell cycle synchronization in optimizing HDR and reducing off-target effects, providing a robust and effective method for human genome engineering.