Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Abstract

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces

“…Research on genes has also progressed in the field of veterinary science, and genetic testing is now commonly used to assess the risks of certain diseases and for drug selection of mast cell tumors in dogs ( Giantin et al, 2012 ; Gil, 2015 ; Vozdova et al, 2019 ). Particularly in equine science, genetic testing is increasingly used for parent-child identification, individual identification, and doping determination in racehorses ( Holl et al, 2017 ; Tozaki et al, 2022 ; 2023 ). However, the collection of genes from animals that do not provide consent sometimes poses problems from an animal welfare perspective.…”

Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses

“…Therefore, the results of our study we consider to contribute to gene-doping control. Recently, we developed a gene-editing test to detect these racehorses [ 14 ]. This test uses the following criterion to identify artificial editing: the presence of homologues of Alt-type INDELs that were not shown in current Thoroughbred populations.…”

“…Recently, a gene-editing test was developed to detect artificially edited sequences using the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) [ 14 ]. This test defined homologous insertions or deletions of novel variants as a type of artificial modification.…”

Long‐read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population

“…Gene editing or disease mutation detection by surveying entire endogenous genes (including introns and regulatory regions), typically requires either whole genome sequencing (Liu et al, 2019), or a targeted panel of hybrid-capture enrichment probes (Lord et al, 2019) or overlapping amplicons (McCabe et al, 2019). A paper describing the use of an amplicon-based targeted panel for gene editing detection in horses has recently been published (Tozaki et al, 2022).…”