Figure 5.

Genome editing in sheep zygotes—use of ICE for screening and validation of gRNA designs and genotyping of edited embryos. (A) Work flow for BCO2 gene editing in Tan sheep, indicating the use of ICE to validate gRNA designs in fibroblasts and genotype embryos prior to generation of lambs. (B) The predominant 1-bp insertion often observed after single gRNA editing is indicated by black arrow. The major 54-bp deletion resulting from dual gRNA editing is indicated by red arrow. (C) Sequence chromatograms from the dual gRNA-edited and control fibroblasts. Note the composite sequence trace from the edited sample as opposed to the single trace from the control sample. (D) List of sequences from the dual gRNA-edited fibroblasts, as displayed by ICE. Note that ICE does not provide the full sequence of indels, which must be deduced by inspecting the boundaries. Note that the list provides indel size, frequency and sequence of the various alleles, although some nucleotide identities are not defined (indicated by ‘N’). (E) ICE indel profiles for three embryos edited with dual gRNAs. The major 54-bp deletion is indicated by red arrow and the low frequency 4-bp deletion by an asterisk.