One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system

Area of Science:

• Genetics and Genomics
• Animal Models
• Neuroscience

Background:

• Pigs possess anatomical and physiological similarities to humans, making them suitable models for human diseases.
• Neurodegenerative diseases, such as Parkinson's disease, are complex and require advanced models for effective study.
• CRISPR/Cas9 gene editing technology offers unprecedented potential for creating precise animal models.

Purpose of the Study:

• To generate a novel pig model for human Parkinson's disease.
• To simultaneously target three key genes (parkin, DJ-1, PINK1) associated with Parkinson's disease using CRISPR/Cas9.
• To assess the efficiency and safety of multiplex gene editing in pigs.

Main Methods:

• Simultaneous targeting of parkin, DJ-1, and PINK1 genes in Bama miniature pigs using CRISPR/Cas9.
• Co-injection of Cas9 mRNA and multiplex single-guide RNAs (sgRNAs) into pronuclear embryos.
• Whole-genome sequencing analysis to evaluate off-target mutations.

Main Results:

• Successfully generated gene-modified piglets targeting three distinct genomic loci.
• The resulting piglets remained healthy and exhibited normal behavior up to 10 months of age.
• Trio-based whole-genome sequencing revealed a low incidence of off-target mutations despite multiplex gene editing.

Conclusions:

• The CRISPR/Cas9 system is highly effective for multiplex gene modification in pigs.
• This study demonstrates the feasibility of creating a valuable pig model for Parkinson's disease research.
• The developed model holds significant potential for advancing the understanding and treatment of neurodegenerative diseases.