Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

Wenjun Zhou,Zhen Wang,Yanli Zhang,Kaiping Deng,Mingtian Deng,Yongjie Wan,Feng Wang,Rihong Guo,Qing-Yuan Sun

doi:10.1371/journal.pone.0186056

Abstract

Goat’s milk, considered a substitute for cow’s milk, has a high nutritional value. However, goat’s milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%–9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

Highlights

The goat (Capra hirus) is one of the most important livestock species, providing products such as meat, hides, and milk
To test the genome editing efficiencies of different transfection methods, a 9-kb plasmid containing enhanced green fluorescent protein was transfected into goat fibroblasts using lipofection or electroporation method
To test the editing efficiency of the three small guide RNAs (sgRNAs), each of the three Cas9-sgRNA plasmids was transfected into goat fibroblasts, and genomic DNA was extracted 72 h after transfection

Summary

Introduction

The goat (Capra hirus) is one of the most important livestock species, providing products such as meat, hides, and milk. Goat’s milk and its byproducts, such as yogurt, cheese, and powder, are important components of the daily human diet in many countries [1]. Goats are used as mammary gland bioreactors in biomedical studies [2]. Goat’s milk has similar nutritional value to cow’s milk, with high percentages of fats and proteins. Β-lactoglobulin (BLG) is a major whey protein allergen in goat and other ruminants’ milk [3]. Hydrolysis and heat do not suppress the allergenicity of BLG, and fermentation byproducts increases its immuno-reactivity [4, 5].

Methods

Results

Conclusion