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

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.

In the last 17 years, horse cloning has focused on multiplying valuable individuals mainly because of commercial interests 1 , Kheiron S.A (www.kheir on-biote ch.com), ViaGen (www.viage n.com)]. The strongest advantage of this technique is the genome conservative property, so that the cloned foals invariably "inherit" the original genotype. However, this technique can be even more powerful when it is combined with gene editing, to obtain horses with the genetic background of the original individual and new desired characteristics in one generation and in a non-random way.
The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of the techniques for gene editing that has successfully been used to improve animal features and to obtain desired genotypes in different species. This system was first described in prokaryotes as an acquired immune system against plasmids and phages 2,3 . Once discovered, this natural system was adapted to become a tool for gene editing in kingdoms as distant as fungi, plants and animals [4][5][6] . By this technique, numerous genome edited animals from different species have been generated 7 . These animals were modified to be potentially immune to infective diseases 8 , to serve as bioreactors 9 , for human disease modeling 10,11 and for xenotransplantation 12,13 .
The main interest in genetically modified horses focuses on disease resistance, genetic disease reversion and sportive performance improvement. Recently, a deleterious mutation in the GBE 1 gene that causes an autosomal recessive condition known as glycogen branching enzyme deficiency and a single nucleotide mutation in the PPIB gene that causes a skin disease in horses were edited in horse fibroblasts by homologous recombination with CRISPR/Cas9 14,15 . In both cases, the normal genotype was restored with a view to later using these cells for cloning. However, no reports on CRISPR/Cas9 application to sportive performance are available so far and targeting the myostatin gene (MSTN) constitutes one of the most promising approaches. www.nature.com/scientificreports/ MSTN is a negative regulator of muscle growth and differentiation 16 . It is expressed in skeletal muscle 17 and mutations in its sequence result in augmented muscle mass. Natural MSTN gene mutations with this phenotype are present in cattle breeds such as the Belgian Blue and Piedmontese 18 , and in dogs 19 . This gene has also been experimentally edited using CRISPR in different mammals including pigs 20,21 , dogs 22 , rabbits 23 , goats 24,25 and sheep 26 , and using TALEN in cattle and sheep 27 . Most of these studies used zygote microinjection to generate the MSTN-knock out (MSTN-KO) genotype. However, this approach may produce mosaic embryos, which may make it difficult to anticipate the mutations generated and/or putative off-targets (OTs) before animal birth, as it would require embryo biopsy followed by whole genome amplification to analyze the genotype of the edited embryo. For these reasons, cloning is postulated as a promising technique to generate gene edited animals as the cell line with the desired edition can be selected before the somatic cell nuclear transfer (SCNT) procedure. This technique becomes even more relevant in species such as the horse, in which embryo generation by intracytoplasmic sperm injection (ICSI) is inefficient 28,29 , and the successful of embryo generation by in vitro fertilization (IVF) is not reliable 30,31 .In this work we aimed to generate edited horse embryos by knocking out MSTN using CRISPR/Cas9 and SCNT (Fig. 1). To our knowledge, this is the first time that edited horse embryos have been generated. This strategy allowed us to choose those clonal cell lines with mono-or bi-allelic editions and no off-targets to be used as nuclear donors for cloning.

Results
Horse fetal fibroblasts nucleofection efficiency. In order to estimate the nucleofection efficiency of horse fetal fibroblasts (HFFs) using the NEON system, we compared different concentrations of the EGFP-N1 plasmid. With this experiment, EGFP expression above 87% was observed by flow cytometry in all conditions (Supplementary Figure S1).
Edition efficiency was dependent on the gRNA and the plasmid concentration. Two gRNAs were designed to target exon 1 of equine MSTN (Fig. 2). The edition efficiency of each gRNA was first evaluated in HFF puromycin resistant cells (cell pools) by PCR amplification (Table 1) of the target region followed by Sanger sequencing. Both gRNAs were able to generate editions in the first exon of MSTN. Moreover, different concentrations of the plasmid were used for nucleofection. According to insertions and deletions (InDel) analysis by Synthego`s Inference of CRISPR Edits (ICE) tool 32 , edition efficiencies were 73%, 93% and 96% for gRNA1 and 88%, 89% and 94% for gRNA2 in cell pools when 1, 2 and 5 µg per 1 × 10 6 cells were used, respectively (Fig. 3A). Therefore, increasing plasmid concentration improved edition efficiency. To further characterize both gRNAs, InDel characteristics were evaluated in the isolated clonal cell lines. We obtained different genotypes depending on the gRNA and the plasmid concentration used (Fig. 3B and Supplementary Table S1). The best Putative off-targets evaluation in cell pools and clonal cell lines. One of the advantages of edited embryo generation by SCNT is the possibility of characterizing the donor cell lines for the gene edition and the absence of OTs. First, we analyzed two high rank putative off-targets (OTs) [according to Benchling online software (https ://www.bench ling.com)] of each gRNA in the six experimental cell pools. After InDel analysis, OTs were only observed in the OT1 of gRNA1-5 µg cell pool, but not in the other 5 experimental groups (Supplementary Figure S2). As OTs accounting for less than 5% may go under detected in a cell pool by Sanger sequencing, the edited clonal cell lines chosen for embryo generation (described below) (Fig. 4) were also subjected to OTs evaluation, revealing no differences with respect to the wild-type control (Fig. 5).

MSTN-KO embryo generation by SCNT.
To evaluate the developmental capacity of the embryos produced using edited cells, three clonal cell lines were selected for further characterization and embryo generation by SCNT. The chosen clonal cell lines were G2-1 µg-C02 (with a monoallelic edition), G2-5 µg-C13 (with biallelic heterozygous editions) and G1-1 µg-C23 (with a biallelic homozygous edition). The edited MSTN sequences of each clonal cell line are detailed in Fig. 4, whereas the results of embryo development are summarized in Table 2. The three clonal cell lines were able to generate blastocysts (Fig. 6), although with lower efficiency than mesenchymal stem cells (MSCs) (p < 0.05) and a non-statistically significant tendency to lower efficiency than the wild-type HFF control. In addition, three of the blastocysts generated were evaluated for MSTN edition and putative OTs, with results showing the same MSTN sequence in the embryos and the clonal cell lines, without OTs (Fig. 5).  www.nature.com/scientificreports/

Discussion
MSTN has been studied and edited in different mammalians' species mainly with the purpose of increasing meat production in cattle 27 , goats 24,25 , sheep 26,27 and pigs 20,21 , and for enhancing sport performance in dogs 22 . In addition, some cattle and dog breeds have natural MSTN loss of function mutations 18,19 . In horses, such mutations have been neither described nor generated, although one single nucleotide polymorphism (SNP) in the MSTN intron sequence has been identified 33 . This SNP has been associated with fitness at different racing distance ranges and with muscle fiber proportions [33][34][35][36] . However, it was later demonstrated that this SNP was linked to a short interspersed nuclear element insertion (SINE insertion) in the MSTN gene promoter 36,37 , which alters the transcription start site and, consequently, the transcript levels of the gene 37 . It has been shown that MSTN expression levels affects the performance ability of each individual, giving faster horses for shorter distances with lower MSTN expression 38 . On the basis of these studies, we decided to edit MSTN to generate KO horse embryos by SCNT and consider other point editions in the future that could enhance sport performance in horses. First, we evaluated two gRNAs targeting the first exon of MSTN. We observed high nucleofection efficiency in HFFs with the EGFP-N1 plasmid using the Neon system and we determined that both gRNAs were suitable for generating InDels in the gene, albeit with different efficiencies. According to InDels analysis (ICE, Synthego) 32 of the cell pools and considering the three different concentrations evaluated, the average efficiencies were 87.33% for gRNA 1 and 90.33% for gRNA2. In addition to gRNA efficiencies, the three experimental concentrations of the plasmid displayed different capabilities to generate InDels in exon1 of MSTN. Both in cell pools and in the individual analysis of clonal cell lines, more editions were observed as the concentration of the plasmid increased. However, high plasmid concentration induced undesired OTs in G1-5 µg conditions and insertions corresponding to plasmid DNA delivered-edition in four clonal cell lines. These kind of insertions were previously described in a hornless genome-edited bull generated by TALEN and a plasmid HDR-donor sequence after whole genome sequencing analysis 39,40 . In addition, monoallelic editions were obtained only when the lowest plasmid concentration was used (1 µg per 1 × 10 6 cells), and higher proportion of biallelic editions were identified in clonal cell lines with higher concentrations of the CRISPR system. Similar results have been reported in pig embryos when different concentrations of Cas9 protein and gRNA were used for zygote microinjection 41 . These results www.nature.com/scientificreports/ strongly suggest that the concentration of CRISPR/Cas9 plasmid or ribonucleoprotein complex directly affects gene editing efficiency and it could be used as a methodological strategy to generate mono-or biallelic editions. Reproductive biotechnology applied to the generation of edited embryos also affects the overall efficiency. Until now, no reliable protocols have been made available for IVF in horses and an efficient method to edit embryos by ICSI has not been yet developed. Moreover, zygote microinjection has proven to have high rates of mosaic embryos/animals 8,42-46 , OT occurrence 26,47 and low birth rates of edited animals 48,49 . In sheep, for example, the reported efficiencies to disrupt the MSTN gene using CRISPR/Cas9 by zygote microinjection were 5.7% 48 , 45.4% 26 and 27.7% 49 . Therefore, we chose the cloning technique as it allows the analysis of the edited gene sequence and putative OT activity prior to the generation of the embryos 50,51 . In this work, we confirmed the absence of two high ranked putative OTs in five of the six cell pools and in the clonal cell lines used to generate the edited embryos.
Despite the great advantages of cloning, one of its disadvantages is the need to generate clonal cell lines, which requires increasing cell passages during the isolation and expansion process. In horses, the rate of nuclear remodeling decreases significantly after embryo reconstruction using fetal fibroblasts of increased passage number 52 . Then, in addition to the low blastocyst rates reported for horse cloning, this factor might explain the lower embryo developmental rate of the edited cloned embryos compared to controls. To increase horse cloning development, MSCs could be used as nuclear donors 1 . However, it was demonstrated that MSCs undergo senescence at early passages, showing alterations in cellular morphology, telomere shortening and proliferation arrest after 30 population doublings or 7-10 passages 53 , which makes the generation of edited clonal cell lines rather difficult. www.nature.com/scientificreports/ In summary, we demonstrate that it is possible to edit HFFs by CRISPR/Cas9 with high efficiency and generate embryos with genetic modifications at the blastocyst stage by SCNT. To our knowledge, edited horse embryos had not been reported until now. With this technique available other editions could be achieved, including the correction of genetic defects that cause equine diseases 15,54 . Our long-term goal is then to identify natural sportadvantageous allele sequences present in the genome of some individuals and incorporate them in others to endow them with the desired characteristics. In this way, we could introduce the SINE insertion in the MSTN promoter of those animals lacking it, in order to alter the proportion of muscle fibers and obtain faster horses for short distances. We consider this a precision breeding strategy which can be achieved in only one generation.

Methods gRNAs design and construction.
Two gRNAs complementary to the first exon of the equine MSTN gene were designed using Benchling 2018 (https ://www.bench ling.com) (Fig. 2). The sequences were gRNA1: TGA TCA ATC AGT TCC CGG AG (chr18:66609845-66609864, EquCab3.0) and gRNA2: TGA TGA TTA CCA CGC GAC GA (chr18:66609780-66609799, EquCab3.0). To obtain synthetic oligonucleotides codifying each gRNA, two complementary oligo DNAs were synthesized, annealed and cloned as previously described 55 . The backbone  Table 1. MSTN primers were designed so that a 654 bp band was obtained with the gRNA1 edition site at 225 bp from the forward primer and the gRNA2 edition site at 303 bp from the forward primer. OT loci were selected according to the OT ranking of Benchling online software (https ://www. bench ling.com). OT specifications are summarized in Table 3. Once the DNA band was confirmed, the PCR product was used for reamplification by PCR, ethanol precipitation, isopropyl alcohol extraction and Sanger Figure 6. MSTN knock-out horse embryos. Three day 7 horse embryos obtained from G1-1 µg-C23 experimental group. Generation of MSTN edited embryos by SCNT. Three edited cell lines were selected as nuclear donors for horse cloning. Two of them were MSTN-KO lines, one with the same edition in each allele (clone G1-1 µg-C23) and the other one with different editions in each allele (clone G2-5 µg-C13). The third cell line used was a heterozygous cell line with one edited allele (clone G2-1 µg-C02) (Fig. 4). Embryo generation by zona free nuclear transfer was performed as previously described by our group 58 . Briefly, ovaries were obtained from local slaughterhouses (Raul Aimar S.A., Ruta 36 km. 597, Río Cuarto, Córdoba, Argentina, ZIP code: 5805) and oocytes were matured for 24 h. Matured oocytes were then treated with pronase (#P-8811, Sigma Aldrich Co., USA) to remove the zona pellucida, enucleated by micromanipulation and electrically fused with a donor cell (wild type fibroblast, G1-1 µg-C23, G2-5 µg-C13 or G2-1 µg-C02 cell). In order to incorporate a positive control, we performed the same procedure using MSCs (with the same genomic background as the HFFs) as nuclear donors, considering the enhanced effectiveness of using this type of cells in horse cloning 1 . After 2.5 h fusion, reconstructed embryos were activated with 8.7 μM ionomycin (#I24222; Invitrogen, CA, USA) for 4 min followed by individual culture in a combination of 1 mM 6-dimethylaminopurine (6-DMAP; # D2629, Sigma Aldrich Co., MO, USA) and 5 mg/ml cycloheximide (CHX; #C7698, Sigma Aldrich Co., MO, USA) in 5 µl drops of DMEM/F12 (#D8062, Gibco, Grand Island, NY, USA) for 4 h. Reconstructed embryos were cultured in DMEM/F12 containing 10% FBS, and 1% penicillin-streptomycin in the Well-of-the-Well system, three embryos together per well. Two experimental replicates were performed with the MSTN edited cells (G1-1 µg-C23, G2-5 µg-C13 or G2-1 µg-C02) together with the wild type HFF control group in each procedure, and one replicate was performed comparing SCNT efficiency with MSCs and HFF wild type donor cells. Finally, embryo development was assessed on day 2 (cleavage rates) and on day 7 (blastocyst rates). www.nature.com/scientificreports/