Fig. 5

Gene engineering using Cas9-expressing germ cells via viral and non-viral methods. A Schematic experiment design for testing sgRNA for PRNP gene knockout utilizing viral and nonviral approaches without introducing Cas9 during the pre-embryonic in vitro culture process. B An SB transposon vector containing both GFP and sgPRNP sequences was microinjected, resulting in mutations identified in embryos that expressed both RFP and GFP (yellow arrow: R + and G + , white arrow: R- and G +). C T7E1 assay result confirming PRNP mutations (Lanes: M: marker, WT: wild type, MI: microinjection, R + : RFP positive, G + : GFP positive, R-: RFP negative, NC: negative control, and PC: positive control). D and E The viral method for gene editing was carried out using AAV6 containing an sgPRNP sequence. T7E1 assay results of PRNP mutation in the RFP-positive group cultured for 72 h after media treatment with AAV6. F Schematic experiment design for knock-in strategy at the BSA gene locus in embryos. G Observation of GFP-positive blastocysts post-AAV6 infection. H Schematic of the knock-in strategy and validation. Diagram showing targeting site on Bos taurus chromosome 6, where sgRNA targets bovine serum albumin (BSA). knock-in donor containing Attb-AfIII sequences for homologous recombination, flanked by 500-bp left and right homology arms (LHA and RHA) (a). PCR validation of the knock-in band. Eight randomly selected GFP-positive blastocysts were sampled, and PCR was carried out with primers targeting sequences both inside and outside the knock-in regions (b). I Summary table of the knock-in efficiency (results from three experiments) including number of cumulus-oocyte complexes (COCs), 8-cell stage embryos, total blastocysts (BL), GFP-positive blastocysts (GFP + BL), and percentage of knock-in in sampled blastocysts