Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system

Area of Science:

• Genetics and Genomics
• Cardiovascular Research
• Animal Models

Background:

• Hyperlipidemia and atherosclerosis are significant human health concerns.
• Existing animal models may not fully recapitulate human disease complexity.
• Novel models are needed for advanced translational research.

Purpose of the Study:

• To generate novel rabbit models with targeted large-fragment deletions in the LDL receptor (LDLR) and/or apolipoprotein E (apoE) genes.
• To establish a platform for studying hyperlipidemia and atherosclerosis.
• To investigate the efficacy of CRISPR/Cas9 for complex gene editing in rabbits.

Main Methods:

• Utilized the CRISPR/Cas9 system with multiple single-guide RNAs (sgRNAs) to edit LDLR and apoE genes in rabbit embryos.
• Sequenced founder rabbits to confirm gene modifications, including large fragment deletions.
• Analyzed plasma lipid and lipoprotein profiles, and performed western blotting for apolipoprotein expression.
• Examined aortic tissues using Sudan IV and HE staining to assess atherosclerosis development.

Main Results:

• Successfully generated six knockout (KO) rabbits: four LDLR KO and two LDLR/apoE double-KO.
• Confirmed diverse mutations, including biallelic large fragment deletions in the LDLR gene.
• Observed significant hyperlipidemia in KO rabbits, with total cholesterol up to 10-fold higher than wild-type.
• Demonstrated prominent aortic and coronary atherosclerosis in founder KO rabbits.

Conclusions:

• CRISPR/Cas9 with multiple sgRNAs is effective for creating large fragment gene deletions in rabbits.
• The generated LDLR KO and LDLR/apoE double-KO rabbits represent valuable new models.
• These models offer a novel approach for translational research into human hyperlipidemia and atherosclerosis.