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 (GBE1102C>A). 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 GBE1102C>A 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.
Highlights
Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations
In order to correct the mutation in a heterozygous American Quarter Horse stallion (Fig. 1), we first established a protocol for clonal isolation after transfection and enrichment by fluorescence-activated cell sorting (FACS)
Karyotypically normal primary fibroblasts derived from the heterozygous stallion (Fig. 2A) were transfected (Fig. 2B,C) and sorted by FACS for viable cells containing the construct (Fig. 2D)
Summary
Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. Precise editing of a genome locus has historically been hampered by the overall low frequencies of homologous recombination, in which successful modification is achieved in one per a thousand transfected cells (0.001%)[13] This changed in 1983 with the discovery that a site-specific double-stranded break (DSB) in genomic DNA stimulates a particular locus to exchange genetic information with either a homologous chromosome or an exogenously provided repair template[14]. Since this discovery, the field of genome editing has pursued the development of technologies to induce site-specific DSBs using engineered nucleases[15]: first with the zinc-finger nucleases (ZFNs)[8,16], transcription activator-like effector nucleases (TALENs)[17,18], and more recently using the CRISPR-Cas system[19,20,21]. DSBs can be repaired by homologous recombination (HR), which is comprised of at least three sub-pathways: homology-directed repair (HDR), single-stranded annealing (SSA) and the recently recognized single-stranded template repair (SSTR)[26,27]
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