Developmental competence and mutation efficiency on microinjection mediated gene editing
To determine optimal conditions, including Cas9 mRNA, and sgRNA concentrations for producing mutations in bovine blastocysts, four experimental conditions were used, then developmental competence and mutation efficiency were investigated in the blastocyst stage following each condition (Fig 1C, D, and E). There was no significant difference in cleavage rate across all 4 groups (data not shown). However, in the RNA4X group a diminished blastocyst formation rate was observed in comparison to the control group (Fig 1D). In terms of mutation rate, the RNA2X group (81.3 ± 17.2%) had the highest mutation compared the other 3 groups (WT: 0%, RNA1X: 33.3 ± 16.0%, RNA4X: 50.0 ± 28.5%) (Fig 1E). Thus, the RNA2X condition was selected as the optimal condition for the further production of MSTN mutated cattle.