This study investigates the use of CRISPR/Cas9-mediated gene editing in human tripronuclear (3PN) zygotes to modify the β-globin gene (HBB). The researchers found that CRISPR/Cas9 could effectively cleave the HBB gene, but the efficiency of homologous recombination-directed repair (HDR) was low, and the edited embryos were mosaic. Off-target cleavage was also observed, as revealed by the T7E1 assay and whole-exome sequencing. Additionally, the endogenous delta-globin gene (HBD) competed with exogenous donor oligos as a repair template, leading to unwanted mutations. The data suggest that HDR of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. The study highlights the need to improve the fidelity and specificity of the CRISPR/Cas9 platform before considering clinical applications.This study investigates the use of CRISPR/Cas9-mediated gene editing in human tripronuclear (3PN) zygotes to modify the β-globin gene (HBB). The researchers found that CRISPR/Cas9 could effectively cleave the HBB gene, but the efficiency of homologous recombination-directed repair (HDR) was low, and the edited embryos were mosaic. Off-target cleavage was also observed, as revealed by the T7E1 assay and whole-exome sequencing. Additionally, the endogenous delta-globin gene (HBD) competed with exogenous donor oligos as a repair template, leading to unwanted mutations. The data suggest that HDR of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. The study highlights the need to improve the fidelity and specificity of the CRISPR/Cas9 platform before considering clinical applications.