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

Xianlong Wang,Hongyong Zhang,Xiao Wang,Guosong Qin,Jiaojiao Huang,Tang Hai,Jinbo Cheng,Yanfang Wang,Jianguo Zhao,Jing Yao,Zengqiang Yuan,Qi Zhou,Hongmei Wang,Qiantao Zheng,Chunwei Cao

doi:10.1038/srep20620

Abstract

Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson’s disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value.

Highlights

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder in the elderly
Several previous studies have reported that simultaneous use of dual single guide RNAs (sgRNAs) to target an individual gene significantly improved the CRISPR/Cas9-mediated genome targeting efficiency[13,14,22]
We found that dual gRNAs could simultaneously target three distinct genomic loci in Bama miniature pigs in a highly efficient manner

Summary

Introduction

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder in the elderly. Through the use of the CRISPR/Cas[9] system, biallelic gene knockout pigs were efficiently generated in a single step through a direct cytoplasmic injection of Cas[9] mRNA and sgRNA into pig zygotes[9,10]. These findings indicated that the CRISPR/Cas[9] system shows enormous potential in establishing large animal models of neurodegenerative diseases by engineering genome in spite of the lack of embryonic stem cell lines for genomic manipulation[11]. It drives us to recapitulate some key features of PD disease by generating DJ-1/parkin/PINK1 triple-gene modified pigs using CRISPR/Cas[9] system

Methods

Results

Conclusion