Abstract
ObjectiveTo generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.MethodsCRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.ResultsSix knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.ConclusionLarge fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.
Highlights
Hyperlipidemia is the major risk factor of atherosclerosis [1]
We attempted to create double-double KO rabbits using CRISPR/Cas9 technology by aiming at large gene fragment deletions via multiple sgRNAs to edit low-density lipoprotein (LDL) receptor (LDLR) and apoE genes. Those KO rabbits with large-fragment LDLR or LDLR/ apoE gene deletions exhibited remarkable hyperlipidemia and developed aortic and coronary atherosclerosis on a normal chow diet. These results indicate that the CRISPR/Cas9 system-directed by multiple sgRNAs-can induce large fragment deletions of the LDLR gene in rabbits and that these LDLR KO and LDLR/apoE double-KO rabbits should provide novel models for elucidating the mechanisms and therapeutic interventions for hyperlipidemia and atherosclerosis
Because our previous article [10] introduced the arterial lesions of LDLR KO rabbits, currently, we focused on the 7♂ — with a large fragment deletion and accompanying severe hyperlipidemia —and the 5♀ rabbit with LDLR/apoE double- KO
Summary
Hyperlipidemia is the major risk factor of atherosclerosis [1]. To study the pathogenesis of atherosclerosis and develop new therapeutics, experimental animal models are essential. We attempted to create double-double KO rabbits using CRISPR/Cas technology by aiming at large gene fragment deletions via multiple sgRNAs to edit LDLR and apoE genes Those KO rabbits with large-fragment LDLR or LDLR/ apoE gene deletions exhibited remarkable hyperlipidemia and developed aortic and coronary atherosclerosis on a normal chow diet. These results indicate that the CRISPR/Cas system-directed by multiple sgRNAs-can induce large fragment deletions of the LDLR gene in rabbits and that these LDLR KO and LDLR/apoE double-KO rabbits should provide novel models for elucidating the mechanisms and therapeutic interventions for hyperlipidemia and atherosclerosis
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