MSTN-edited born calves obtained by precision breeding, using CRISPR/Cas9 and SCNT

Abstract

Abstract Gene editing in cattle offers great potential in the livestock industry. To enhance beef productivity, the aim of this study was to obtain MSTN-edited calves by combining CRISPR/Cas9 edition in cell culture with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), gene editing efficiency was evaluated using plasmid-based CRISPR/Cas9 edition in fetal fibroblasts (BFF-E1 cells). The bioinformatic predicted edition rate (BPE) in BFF-E1 was 96%, and all cloned blastocysts generated using these cells as nuclear donors presented bi-allelic edition. In a second experiment (E2), Cas9 protein and trac:crRNA oligoribonucleotide (RNP) were used for MSTN gene editing of one fetal fibroblast (BFF-E2-male) and two mesenchymal stem cell lines (MSC-E2-male and MSC-E2-fem) from price-winning animals. The BPEs were 58.8%, 31% and 59% in cells, and 64%, 73.3% and 66.6% in SCNT embryos, respectively. Heterozygous and wild-type embryos were obtained in all E2 groups. One MSTN-edited calf was born from MSC-E2-femed group. Sequencing analysis revealed heterozygous biallelic edition in exon 2, consisting of an insertion of a thymine (T) base, and a deletion of 18 nucleotides (MSTNKO/-6). A second generation MSTNKO/-6 cloned calf was obtained. In conclusion, high rates of edited blastocysts with valuable genetic background and the birth of two edited calves for the MSTN gene were achieved through RNP-based editing. The protocol described in this work establishes the basis to induce gene editions with productive or biomedical relevance.