Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system

Abstract

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 with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…In most cases, the studies have chosen to target the first exon of the gene and obtained small deletions and insertions in the gene. In the studies of Proudfoot et al (2015), Han et al (2014), Luo et al (2014), Crispo et al (2015), Wang et al (2015c), and Wang et al (2015a) several offspring were born that had the desired mstn knock-out. The animals were visually different from the wild type individuals with an obvious difference in muscle mass and a difference was also observed during histological comparison of the muscles Qian et al, 2015).…”

“…The knock-out of the mstn gene thus results in a substantial increase of skeletal muscle mass and in increased meat production. Using SDNs, the gene was artificially mutated in cattle Proudfoot et al, 2015) and pigs (Huang et al, 2014b;Qian et al, 2015;Wang et al, 2015b), but also in smaller livestock species like sheep Zhang et al, 2014a;Crispo et al, 2015;Proudfoot et al, 2015) and goat (Ni et al, 2014;Wang et al, 2015c). In most cases, the studies have chosen to target the first exon of the gene and obtained small deletions and insertions in the gene.…”

Intracytoplasmic sperm injection in domestic and wild mammals

“…Moreover, after the generation of the first transgenic piglet by sperm injection, ICSI gained importance as a new tool for inducing genetic modifications in farm animals (Kurome et al 2006, Garcia-Vazquez et al 2010. However, the advent of CRISPR-Cas9 system for genetic engineering lead to the replacement of ICSI by regular or IVF zygotes for the generation of genetically modified animals (Hai et al 2014, Whitworth et al 2014, Proudfoot et al 2015, Wang et al 2015, Bevacqua et al 2016.…”

“…In conclusion, ICSI-MGT in farm animal has only produced repeatable results in pigs, wherein several transgenic offspring have resulted. However, interest in producing transgenic animals by ICSI-MGT has decreased with the advent of new tools of gene edition (CRISPR-Cas9 and TALEN system), which are technically simpler (Hai et al 2014, Whitworth et al 2014, Proudfoot et al 2015, Wang et al 2015, Bevacqua et al 2016). …”

Research progress of gene editing technology CRISPR/Cas9 system in animal gene editing

“…After both double gene modifi cation, and the result showed 10% of animals survived. These studies suggest that the CRISPR/Cas9 system can be used as an effective gene editing tool for breeding new varieties of animal traits and breeding for disease resistance [34]. By injecting the Cas9 mRNA and sgRNA of the IL2RG and Rag1 genes into the cytoplasm of prokaryotic embryos, bi-allelic knockout rabbits can be obtained and the effi ciency up to 100%, while knocking out 3 genes (IL2RG, RAG1, and RAG2) and 5 genes (IL2RG, RAG1, RAG2, TIKI1 and ALB), the effi ciency could reach 33.3% [35].…”