CRISPR/Cas9-Mediated Generation of Guangxi Bama Minipigs Harboring Three Mutations in α-Synuclein Causing Parkinson’s Disease

Parkinson’s disease (PD) is a common, progressive neurodegenerative disorder characterized by classical motor dysfunction and is associated with α-synuclein-immunopositive pathology and the loss of dopaminergic neurons in the substantia nigra (SN). Several missense mutations in the α-synuclein gene SCNA have been identified as cause of inherited PD, providing a practical strategy to generate genetically modified animal models for PD research. Since minipigs share many physiological and anatomical similarities to humans, we proposed that genetically modified minipigs carrying PD-causing mutations can serve as an ideal model for PD research. In the present study, we attempted to model PD by generating Guangxi Bama minipigs with three PD-causing missense mutations (E46K, H50Q and G51D) in SCNA using CRISPR/Cas9-mediated gene editing combining with somatic cell nuclear transfer (SCNT) technique. We successfully generated a total of eight SCNT-derived Guangxi Bama minipigs with the desired heterozygous SCNA mutations integrated into genome, and we also confirmed by DNA sequencing that these minipigs expressed mutant α-synuclein at the transcription level. However, immunohistochemical analysis was not able to detect PD-specific pathological changes such as α-synuclein-immunopositive pathology and loss of SN dopaminergic neurons in the gene-edited minipigs at 3 months of age. In summary, we successfully generated Guangxi Bama minipigs harboring three PD-casusing mutations (E46K, H50Q and G51D) in SCNA. As they continue to develop, these gene editing minipigs need to be regularly teseted for the presence of PD-like pathological features in order to validate the use of this large-animal model in PD research.