Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.
Sci Rep
; 10(1): 7411, 2020 05 04.
Article
en En
| MEDLINE
| ID: mdl-32366884
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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Enfermedad del Almacenamiento de Glucógeno Tipo IV
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Exones
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Mutación Puntual
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Fibroblastos
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Sistemas CRISPR-Cas
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Edición Génica
Idioma:
En
Revista:
Sci Rep
Año:
2020
Tipo del documento:
Article