Heritable Gene Disruption in Goats with CRISPR/Cas9 Results in Expected Phenotypes

Precision genetic engineering will accelerate the genetic improvement of livestock for agriculture and biomedicine. 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, we successfully generated gene-modified goats with either one or both genes disrupted through co-injection of one-cell-stage embryos with Cas9 mRNA and sgRNAs targeting two well-known genes MSTN and FGF5. 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 targeting two genes simultaneously was 10%. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder animals, were carefully analyzed, indicating that simultaneous editing of several sites can be achieved in large animals. We further show that the utility of the CRISPR/Cas9 system by disrupting MSTN and FGF5 resulting in expected phenotypes, for instance, higher body weight in MSTN-disrupted animals and increased fiber length in FGF5-disurpted animals. We provided adequate evidence to illustrate that the gene modifications induced by the disruption of FGF5 did occurred at both morphological and genetic levels. In addition, the knockout alleles were capable of germline transmitted. Together with studies carried out in other gene-modified livestock species such as pigs, our results demonstrate 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 to breeding purposes.