Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

Abstract

The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

Figure 1

Experimental design for the generation of MSTN-KO cloned embryos in the horse. Horse fetal fibroblasts were nucleofected with different concentrations of the CRISPR/Cas9 system containing two gRNAs (gRNA1 and gRNA2) directed to the first exon of the myostatin gene (MSTN). After puromycin selection, the surviving cells (cell pools) for each condition were subjected to clonal culture and subsequently sequenced for MSTN. Three MSTN edited clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer (SCNT): one clonal cell line with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition. MSTN edited blastocysts were obtained.

Figure 2

Schematic representation of horse MSTN gene and gRNAs design. The white boxes and lines represent exons and introns, respectively. Inserted grey box represents 5′ untranslated region (5′UTR). The sequence below represents part of exon 1 containing Cas9/gRNA target sites for gRNA1 and gRNA2 labeled in blue. Protospacer-adjacent motif (PAM) is labeled in orange. Black arrows represent transcription start sites.

Figure 3

MSTN gene editions with gRNA1 and gRNA2. (a) Histograms with nucleotide sequence data of cell pools edited with 1, 2 and 5 µg per 1 × 10⁶ cells of CRISPR system. Different edited genotypes predicted by InDels analysis (ICE, Synthego) and percentage of edition for each condition. (b) Ratio of MSTN genotype in cell clones with both gRNAs and different plasmid concentrations. Wt/wt, cell clones with no editions; Wt/ed-X, cell clones with monoallelic editions; ed-X/ed-Y, cell clones with biallelic heterozygous editions; ed-X/ed-X cell clones with biallelic homozygous editions.

Figure 4

MSTN edited sequences of three cell lines chosen for embryo generation by SCNT. Sanger sequencing of three cell lines with different editions. gRNA1 and gRNA2 sequences are shown in blue with the dotted line pointing the cut site of Cas9 for each gRNA. The protospacer-adjacent motif (PAM) is labeled in orange. Both deletions (del) or insertions (ins) are detailed for each cell line.

Figure 5

MSTN and two putative off-target sequences in edited clonal cell lines and blastocysts generated by SCNT. (a) Chromatograms obtained by Sanger sequencing of G2-5 µg-C13 MSTN edition and two putative off-targets (OT1 and OT2) in the clonal cell line and two blastocysts obtained after SCNT with these cells as nuclear donors. (b) Same analysis as in (a) in the G1-1 µg-C23 group, in this case on only one embryo.

Figure 6

MSTN knock-out horse embryos. Three day 7 horse embryos obtained from G1-1 µg-C23 experimental group.