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Scientific reports2020; 10(1); 15587; doi: 10.1038/s41598-020-72040-4

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.
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Publication Date: 2020-09-24 PubMed ID: 32973188PubMed Central: PMC7518276DOI: 10.1038/s41598-020-72040-4Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study focuses on using gene-editing technology, specifically CRISPR/Cas9, to knock out the myostatin gene in horse embryos. This gene negatively impacts muscle mass development. The research showed that higher concentrations of the gene-editing plasmids resulted in more efficient modification and that it’s possible to create horse embryos with the desired gene modifications.

Methodology

  • The researchers used CRISPR/Cas9 technology to target and disrupt the myostatin gene (MSTN). This gene is known to limit muscle growth.
  • Horse fetal fibroblasts (cells that produce collagen and other fibers and are crucial for healing wounds) were treated with two different gRNA/Cas9 plasmids, intending to target the first exon of the MSTN gene.
  • Different concentrations (1, 2, or 5 µg) of these plasmids were used to determine the optimal amount needed for efficient gene editing.

Results

  • It was observed that higher plasmid concentrations resulted in a more efficient gene edition.
  • Specifically, the mutation efficiencies were 63.6% for gRNA1 and 96.2% for gRNA2.
  • The researchers then selected three clonal cell lines, representing three different gene edition outcomes, to produce embryos via a process called somatic cell nuclear transfer, which is a cloning method.
  • All embryos were successfully edited.

Verification & Analysis

  • They analyzed both the MSTN editions and off-targets in the embryos to verify the edits made and evaluate the specificity of the method.
  • The study concluded that CRISPR/Cas9 is a highly efficient and dose-dependent method for the effective modification of the horse’s genome.

Implications

  • The successful application of this technology in horses suggests it could be adapted to create alleles that give sport horses an advantage.
  • This also opens the door for developing precision breeding programs in other industries that could benefit from gene-edited embryos, such as livestock production or biomedical research, which could significantly advance these fields.

Cite This Article

APA
Moro LN, Viale DL, Bastón JI, Arnold V, Suvá M, Wiedenmann E, Olguín M, Miriuka S, Vichera G. (2020). Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer. Sci Rep, 10(1), 15587. https://doi.org/10.1038/s41598-020-72040-4

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 10
Issue: 1
Pages: 15587

Researcher Affiliations

Moro, Lucia Natalia
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina. lmoro@fleni.org.ar.
  • Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. lmoro@fleni.org.ar.
Viale, Diego Luis
  • Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
  • Laboratorio de Neurología y Citogenética Molecular, CESyMA, Buenos Aires, Argentina.
Bastón, Juan Ignacio
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina.
Arnold, Victoria
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina.
Suvá, Mariana
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina.
Wiedenmann, Elisabet
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina.
Olguín, Martín
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina.
Miriuka, Santiago
  • LIAN-CONICET, Fundación FLENI, Buenos Aires, Argentina.
  • Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Vichera, Gabriel
  • KHEIRON BIOTECH S.A, Pilar, Buenos Aires, Argentina. vichera@kheiron-biotech.com.

MeSH Terms

  • Animals
  • Animals, Genetically Modified / genetics
  • Base Sequence
  • CRISPR-Cas Systems
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Female
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Editing
  • Gene Knockout Techniques / veterinary
  • Horses
  • Mutation
  • Myostatin / antagonists & inhibitors
  • Myostatin / genetics
  • Nuclear Transfer Techniques / veterinary
  • Sequence Homology

Conflict of Interest Statement

The authors declare no competing interests.

References

This article includes 58 references

Citations

This article has been cited 19 times.
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