Fig. 4

Utilization of Cas9-expressing somatic cells for gene knockout and embryonic development via somatic cell nuclear transfer (SCNT). A T7E1 assay results showing successful specific gene editing to several genes (PRNP, BLG, Rb1, NANOG, P53, and BCN) using Cas9-expressing somatic cells. (M: Marker, Ctrl: #R2-2 somatic cell as control, KO: Ctrl group with gRNA transfection). B Schematic design of the procedure for creating IFNT mutated blastocysts. Cas9-expressing somatic cells are transfected with sgRNA targeting the IFNT gene for using as a donor cell of SCNT. C IFNT mutation detection of Cas9-expressing single cell colonies transfected with IFNT sgRNA using T7E1 assay(#1–7 = single cell colony) and (D) Sanger sequencing (yellow = sgRNA for IFNT target sequence). E Table showing pre-embryonic developmental competency of cloned embryos from both groups (Control and IFNT KO). F Schematic design of the procedure for creating PRNP mutated blastocysts. The oocyte-fused with Cas9-expressing donor cell is microinjected with sgRNA for PRNP for KO blastocyst production. G PRNP mutation detection of embryo derived from injection of sgRNA targeting PRNP after Cas9-expressing donor cell fusion using the T7E1 assay. H Representative Sanger sequencing result showing a −7 bp deletion in the PRNP target region