Single guide RNA design and testing on bovine fibroblasts
To produce the MSTN mutated cattle, single guide RNA (sgRNA) forMSTN targeting exon2 was designed by CHOPCHOP software (https://chopchop.cbu.uib.no/) that selects sgRNA candidates for target genome (Fig 1A-a). Then the sgRNA for MSTN , and Cas9 protein were co-transfected on bovine fibroblast cells. After 3 days of transfection, they were harvested for extraction of genome DNA. Through the T7E1 assay, it was confirmed whether the target MSTN gene was mutated.