Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0153-5) contains supplementary material, which is available to authorized users.

Summary In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors DAXX and ATRX. Using genome-wide binding and transcriptome analysis, we found that DAXX and ATRX have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wildtype mouse ESCs. Global DNA hypomethylation further promoted recruitment of the DAXX/ATRX complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of DAXX/ATRX in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, DAXX/ATRX-mediated repression seems to involve SUV39H recruitment and H3K9 trimethylation. Our data therefore suggest that DAXX and ATRX safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

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