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Home / Equine Research Bank / Sci Rep / Efficient correction of a deleterious point mutation in prim...
Scientific reports2020; 10(1); 7411; doi: 10.1038/s41598-020-62723-3

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Abstract: Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1 mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.
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Publication Date: 2020-05-04 PubMed ID: 32366884PubMed Central: PMC7198616DOI: 10.1038/s41598-020-62723-3Google Scholar: Lookup
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  • Journal Article

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.

This study used a precise genome editing tool to effectively correct a harmful mutation that causes Glycogen Branching Enzyme Deficiency (GBED) in horses.

Objective of the study

  • The main objective of the research was to find an effective method to correct a harmful genetic mutation responsible for a condition known as Glycogen Branching Enzyme Deficiency (GBED) in horses, which is considered a byproduct of selective breeding during animal domestication. This was to be achieved using a state-of-the-art genome editing tool, CRISPR-Cas9.

Materials and Methods

  • The researchers utilized RNA-guided nuclease CRISPR-Cas9 which is renowned for its precision in genome editing.
  • The experiment was carried out in a primary fibroblast cell line that was derived from a stallion carrying the defective GBE1 gene. This gene contains a genetic mutation (102C > A) which is the main cause of GBED in horses.
  • To rectify this mutation, the researchers designed an assortment of single guide RNAs (sgRNAs) to isolate the mutation, in addition to providing different single-stranded donor DNA templates.
  • The repair process was performed through a method known as homologous recombination (HR).

Findings of the Study

  • Results revealed that the proximity of Cas9-mediated double-strand break (DSB) to the mutation site was more significant in determining the success of the HR process, rather than the efficiency of the DSB.

Conclusion

  • Overall, the study provides an effective approach to correcting specific harmful genetic mutations in horses. The researchers propose that the same principle could potentially be applied to address other harmful diseases in a variety of animal populations.

Cite This Article

APA
Pinzon-Arteaga C, Snyder MD, Lazzarotto CR, Moreno NF, Juras R, Raudsepp T, Golding MC, Varner DD, Long CR. (2020). Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9. Sci Rep, 10(1), 7411. https://doi.org/10.1038/s41598-020-62723-3

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 10
Issue: 1
Pages: 7411
PII: 7411

Researcher Affiliations

Pinzon-Arteaga, Carlos
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.
  • Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Snyder, Matthew D
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.
Lazzarotto, Cicera R
  • University of Fortaleza, Fortaleza-CE, Brazil.
Moreno, Nicolas F
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.
Juras, Rytis
  • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
Raudsepp, Terje
  • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
Golding, Michael C
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA.
Varner, Dickson D
  • Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, USA.
Long, Charles R
  • Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA. clong@cvm.tamu.edu.

MeSH Terms

  • Animals
  • Apoptosis
  • Biotechnology / methods
  • CRISPR-Cas Systems
  • Cell Line
  • Exons
  • Fibroblasts / metabolism
  • Gene Editing
  • Genetic Engineering / methods
  • Glycogen Storage Disease Type IV / genetics
  • Glycogen Storage Disease Type IV / therapy
  • Glycogen Storage Disease Type IV / veterinary
  • Homologous Recombination
  • Horses
  • Karyotyping
  • Phenotype
  • Point Mutation
  • RNA, Guide, Kinetoplastida / genetics
  • Skin / metabolism

Conflict of Interest Statement

The authors declare no competing interests.

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Citations

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