Generation of Quarter- and Hemi-embryos via blastomere isolation reduces mosaicism and reveals editing patterns in Cas9 RNP-edited bovine embryos

Abstract

Mosaicism is a persistent limitation in CRISPR/Cas9-based genome editing of preimplantation embryos. This study tested whether isolating blastomeres at the 2- or 4-cell stage reduces mosaicism in the resulting blastocysts and used the resulting variant patterns at the target region to analyze the duration of activity of Cas9-RNP complexes after electroporation. In vitro-produced bovine zygotes were Cas9-RNP-electroporated against the Tyrosinase (TYR) gene 10 h post insemination and individual blastomeres of cleaved embryos at the 2- or 4-cell stage were isolated to generate Hemi- and Quarter-embryos. While individual culture of embryos did not reduce blastocyst formation compared with group culture, blastocyst yield per source embryo was significantly higher in Hemi-embryos than in zona-free control (88.2 ± 29.0% vs. 51.1 ± 08.1%), whereas the blastocyst yield per source embryo of Quarter-embryos (68.2 ± 28.8%) did not differ significantly from any of the other four treatment groups. While editing efficiency was not affected by zona removal and splitting, mosaicism, however, was significantly reduced in Quarter- and Hemi-embryos versus intact embryos (21.4 ± 10.8% vs. 29.3 ± 31.2% vs. 59.8 ± 32.9%), respectively. Editing event evaluation demonstrated that approximately 66% of presumptive monoallelic edits, if these had been identified in the unsplit embryo, were actually mosaic. Finally, maximum allelic variant counts at the target region were consistent with Cas9-RNP activity persisting at least through S phase associated with the second cleavage division (corresponding approximately to the 4-cell stage) and becoming progressively restricted thereafter. These allelic patterns provide an indirect estimate of the temporal window of detectable Cas9-RNP activity during early embryonic cleavage. In conclusion, the results of the present study infer the temporal window of Cas9 activity during early embryo cleavages. Altogether, blastomere isolation markedly reduced rates of mosaicism offering attractive implications for optimizing genome-editing strategies in livestock breeding.