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

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.

Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9

“…Therefore, the creation of a sCAT line with higher Cas9 expression is desirable. Our most important finding, however, is that this novel gene-editing system based on the use of maCas9 considerably facilitates the generation of mice with single and multiple gene modifications and could be applied to various animal species other than mice [16][17][18][19][20] . In addition, similar effects could be obtained using other Cas proteins 41,42 .…”

Livestock breeding for the 21st century: the promise of the editing revolution

“…The two main genotypes of PRRS virus (PRRSV) share about 60% sequence identity, with further genetic disparity between isolates within each genotype. Control is generally with modified live vaccines, but cross-protection between viral strains is poor [28] . In vitro studies identified CD163, a member of the scavenger receptor cysteine-rich superfamily, as necessary for establishment of a productive viral infection [29] .…”

Targeted Genome Editing for Cotton Improvement

“…Targeted genome modifications have modernized the field of genome engineering and biotechnology by GenEd from unicellular to multicellular and from prokaryotic to eukaryotic organisms. A diversity of organisms from bacteria to humans such as Arabidopsis thaliana [1], tobacco [2], rice [3], yeast (Saccharomyces cerevisiae) [4], fungi [5], zebrafish [6], rats [7], sheep [8], Caenorhabditis elegans [9], human cell lines [10], Drosophila [11], viruses [12][13][14], bacteria [15], mouse [16], insects [17], cattle [18], goat [19], pigs [20], tomato [21], grapes [22], potato [23], soybean [24], maize [25], wheat [26], and cotton [27,28] have been targeted successfully with engineered proteins and nucleases.…”

“…Multiplexing through CRISPR/Cas9 has been used successfully in model and crop plants [19,66,67]. Multiplex genome editing may also be useful for studying functions of gene families as well as an interaction between multiple genes.…”