94 Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning

Some European cattle breeds, such as Charolais and Maine Anjou, have natural mutations in the myostatin gene (MSTN) that inhibit its expression and result in an increase in muscle mass and protein content. An innovative hallmark would be the invitro introduction of this genotype in South America cattle breeds to improve their commercial value. To achieve this, we aimed to disrupt MSTN gene expression in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate MSTN-edited embryos by somatic cell nuclear transfer (SCNT). BFFs were isolated from a cloned fetus of a Brangus bull with a prized genetic background and nucleofected with the Cas9 ribonucleoprotein-gRNA complex previously assessed to target exon 2 of the bovine MSTN gene. To evaluate MSTN editing, genomic DNA from the wild-type (WT) and nucleofected BFFs were isolated, exon 2 of MSTN was amplified by PCR, and the PCR product was Sanger sequenced. In all cases, the sequencing results were analysed using the indel Synthego software tool. According to indel analysis, MSTN gene editing efficiency of the BFFs was 58.83%±3.2 (n=6). The resulting edit consisted of insertion of thymine in exon 2 of the MSTN gene that shifted the gene reading frame, introducing a premature stop codon and generating a truncated MSTN protein. The nucleofected BFFs were then used to generate embryos by SCNT, and WT BFFs were included as controls. Embryo cleavage and blastocyst development rates were evaluated at Day 2 and 7, respectively (Chi-squared test, P<0.05). Although lower cleavage rates were obtained in the MSTN-edited BFFs group [65.3% (n=273/418) vs. 87.6% (n=169/193)], no differences were observed at the blastocyst stage [19.1% (n=80/418) vs. 25.4% (n=49/193)]. To confirm the efficiency of MSTN editing in the cloned embryos, 10 blastocysts generated with the MSTN-edited BFFs were individually analysed for MSTN exon 2 sequence as described before. The results showed that 30% of the blastocysts (3/10) presented a homozygous biallelic edition, which consisted of a thymine base insertion, as expected. In summary, the strategy we used allowed production of MSTN null cloned Brangus embryos avoiding putative undesired integration of exogenous DNA into the bovine genome, such as plasmid sequence, regardless of off-target occurrences. We conclude that CRISPR/Cas9 is an efficient technique to disrupts MSTN gene expression in bovine embryos; this work represents a step toward improving the production efficiency of South American cattle breeds.