Use of an exogenous DNA-free system to generate MSTN-KO calves by CRISPR/Cas9 and SCNT

This study aimed to obtain myostatin (MSTN)-knockout calves, while avoiding the risk of exogenous DNA integration during gene editing. To achieve this, we combined CRISPR/Cas9 ribonucleoprotein gene editing with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), we compared the gene editing efficiency of four gRNAs targeting different coding regions of the MSTN gene using plasmid-based CRISPR/Cas9 in bovine fetal fibroblasts (BFF-E1 cells). The highest bioinformatically-predicted editing rate (BPE) was obtained with gRNA2 (96 %), which was subsequently used for further experiments. Next, embryos were produced by SCNT using BFF-E1-edited cells as nuclear donors. Sanger sequencing of the embryos showed biallelic MSTN editing. In the second experiment (E2), plasmid-based editing was replaced with CAS9 protein and trac:crRNA oligoribonucleotides. Editing efficiency was assessed on one edited bovine fetal fibroblast line (BFF-E2-male^ed) and two edited bovine mesenchymal stem lines (MSC-E2-male^ed and MSC-E2-fem^ed) derived from price-winning animals. BPEs were 58.8 %, 31 % and 59 % in fibroblast and MSC cells, and 64 %, 73.3 %, and 66.6 % in SCNT embryos derived from BFF-E2-male^ed, MSC-E2-male^ed and MSC-E2-fem^ed, respectively. Transfer of 35 MSC-E2-fem^ed embryos to recipient cows, resulted in the birth of one MSTN-edited calf with a heterozygous genotype. A second-generation clone was subsequently produced, using a fibroblast sample as nuclear donor. In conclusion, we established an efficient protocol for generating high rates of edited blastocysts with a desirable genetic background, resulting in the birth of two MSTN-knockout calves. This study provides a foundation for gene editing to improve productive or biomedical traits.