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Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes ( MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed.
The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding. Genome-editing technologies rely on the use of engineered nucleases to induce cellular DNA repair mechanisms and introduce programmable, site-specific genetic modifications in diverse systems. These programmable endonucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently developed, the clustered regularly interspaced short palindromic repeats CRISPR-associated 9 (Cas9) system. The CRISPR/Cas9 system uses short, single-guide RNAs (sgRNA) that recognize the target DNA, then programmed the Cas9 towards targets that are complementary to the first 20 nucleotides of the sgRNA. Compared with ZFNs and TALENs, the RNA-guided CRISPR/Cas9 system demonstrates its precious, versatile and robust merits for targeted genome editing in a variety of species, including model organisms, as well as crops and animals that are crucial to agriculture.
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As one of the first domestic farm animals, the goat is one of the most important livestock species and provides a variety of products, including fiber, milk, meat, and hides. Furthermore, goats have also been used as a model in biomedical studies. Although specific gene knockout strategies based on homologous recombination (HR) and somatic cell nuclear transfer (SCNT) have been established in goats, precise gene modification of the goat genome is still challenging. In a previous study on disruption of four genes simultaneously in goat primary fibroblasts by the CRISPR/Cas9-mediated approach, only the myostatin ( MSTN) knockout fibroblasts were achieved and resulted in live-born goats by SCNT. However, an anti-biotic selection cassette is normally essential for isolating single-cell colonies from seeded donor cells, and reconstructed embryos have a low developmental potential, leading to a relatively low SCNT targeting efficiency (for example, 1–5% in pigs and >10% in cattle). Co-injection of Cas9 mRNA and sgRNA into one-cell stage embryos has been demonstrated to be an efficient approach for the generation of genetically modified mice, rats, monkeys and pigs,,,, which encourages us to extend the application of this strategy to gene targeting in goats. In the present study, through co-injection of one-cell stage embryos of cashmere goats with sgRNAs of two functional genes ( MSTN and FGF5) and Cas9 mRNA, targeted modifications of one or two genes were achieved at an efficiency around 26.5%.
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