This study investigates the efficiency and specificity of CRISPR/Cas9-mediated gene editing in human tripronuclear (3PN) zygotes. The researchers used CRISPR/Cas9 to target the β-globin (HBB) gene, which is associated with β-thalassemia. They found that CRISPR/Cas9 could effectively cleave the HBB gene, but the efficiency of homologous recombination directed repair (HDR) was low, leading to mosaic edited embryos. Off-target cleavage was also observed, as revealed by T7E1 assay and whole-exome sequencing. The endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to unintended mutations. The study also showed that repair of the HBB locus occurred preferentially through the non-crossover HDR pathway. These findings highlight the need to improve the fidelity and specificity of the CRISPR/Cas9 platform for clinical applications. The study also found that CRISPR/Cas9 has off-target effects in human 3PN embryos, and that HDR in human early embryos preferentially occurs through the non-crossover pathway. The study underscores the challenges facing clinical applications of CRISPR/Cas9 due to the potential for off-target effects and the complexity of HDR in human embryos. The research provides important insights into the mechanisms of CRISPR/Cas9-mediated gene editing in human embryos and highlights the need for further investigation to improve the safety and efficacy of this technology.This study investigates the efficiency and specificity of CRISPR/Cas9-mediated gene editing in human tripronuclear (3PN) zygotes. The researchers used CRISPR/Cas9 to target the β-globin (HBB) gene, which is associated with β-thalassemia. They found that CRISPR/Cas9 could effectively cleave the HBB gene, but the efficiency of homologous recombination directed repair (HDR) was low, leading to mosaic edited embryos. Off-target cleavage was also observed, as revealed by T7E1 assay and whole-exome sequencing. The endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to unintended mutations. The study also showed that repair of the HBB locus occurred preferentially through the non-crossover HDR pathway. These findings highlight the need to improve the fidelity and specificity of the CRISPR/Cas9 platform for clinical applications. The study also found that CRISPR/Cas9 has off-target effects in human 3PN embryos, and that HDR in human early embryos preferentially occurs through the non-crossover pathway. The study underscores the challenges facing clinical applications of CRISPR/Cas9 due to the potential for off-target effects and the complexity of HDR in human embryos. The research provides important insights into the mechanisms of CRISPR/Cas9-mediated gene editing in human embryos and highlights the need for further investigation to improve the safety and efficacy of this technology.