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

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.

Gene Editing in Rabbits: Unique Opportunities for Translational Biomedical Research

“…The apoE knockout mouse is a widely used model for the study of human hyperlipidemia. With the CRISPR/Cas9 technology, we and others have generated the apoE knockout rabbits ( Yang et al, 2014 ; Niimi et al, 2016 ; Yuan et al, 2019 ). The homozygous apoE knockout rabbits showed mild hyperlipidemia when fed a normal diet but developed much more severe hyperlipidemia when challenged with a cholesterol-rich diet, compared to the wild-type control rabbits.…”

“…Consequently, the apoE knockout rabbits also developed more significant aortic atherosclerosis than wild-type rabbits upon cholesterol-rich diet challenge for as short as 10 weeks. With multiple sgRNAs targeting rabbit genes, Yuan et al (2019) produced apoE and LDLR double knockout rabbits. This rabbit model can be used to investigate spontaneous hyperlipidemia and atherosclerosis.…”

Rabbit Biomodels of Human Diseases Developed Using New Genomic Technologies. Crispr/Cas9 (Review)

“…При одновременном введении нескольких sgRNA и исследовании 30-ти потенциальных неспецифических мишеней их модификаций не нашли [98]. У KO PFEX кроликов при исследовании 14-ти потенциальных сайтов лишних мутаций не обнаружили [80].…”

“…У КО-кроликов была выраженная гиперлипидемия с уровнем холестерина в 10 раз большим по сравнению с кроликами дикого типа. У КО F0 кроликов развивался аортальный и коронарный атеросклероз [98]. Ожирение и метаболический синдром -проблемы социально-медицинского значения.…”

Development of a rabbit model of Wiskott‐Aldrich syndrome

“…Their physiology, anatomy, and genetic background are more similar to those of humans than those of mice. [17][18][19] Hence, rabbits have been widely applied as reliable models of numerous human diseases, including atherosclerosis, 20,21 Alzheimer's disease, 22,23 arthritis, 24,25 muscle disease, 26 cancers, 27,28 reproductive processes, 29 immune disorders, 30 and infectious disease. 17 Furthermore, owing to their moderate size, low cost, easy breeding, and short gestation period, rabbits can be made extensively available in different laboratories.…”