CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.

Wenni You,Yanbing Wang,Yiwu Chen,Ying Liu,Hongsheng Ouyang,Li Li,Yilin Qi,Daxin Pang,Mengjing Li

doi:10.3390/genes12071027

Abstract

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

Highlights

With the rapid development of biotechnology, genetic modification technology has gradually been applied to animal breeding
The insulin-like growth factor-1 (IGF-1) and Fat-1 genes were cloned into the PUC57 vector, and internal ribosome entry site (IRES) was added upstream of the insulin growth factor (IGF)-1 CDS
To insert the Fat-1 and IGF-1 genes into the porcine Rosa26 site (Figure 1B), the plasmids pX330sgRNA91 (Figure 1C) and PUC57-IRES-IGF1-2A-Fat1-KI were co-transfected into porcine fetal fibroblasts (PFFs) by electroporation

Summary

Introduction

With the rapid development of biotechnology, genetic modification technology has gradually been applied to animal breeding. In the past few years, major achievements have been made in improving the traits of transgenic pigs via the CRISPR/Cas9-mediated knock-in of foreign genes [1,2,3,4,5], providing unprecedented potential for accelerating pig agricultural breeding. Researchers have developed knock-in pigs expressing the pRSAD2 [6], UCP1 [7] and PBD-2 [8] genes. Modified hornless cows were produced by the genomic integration of the POLT gene, thereby avoiding the pain caused by dehorning [10]. Methods for simultaneously inserting multiple foreign genes to improve multiple transgenic animal traits are urgently needed

Methods

Results

Discussion

Conclusion