CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells

Abstract

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…This shows that biPS-1 is suitable for additive and potentially for subtractive genetic modification and may be used for the production of genetically modified cattle via SCNT (Cao et al, 2012;Saito et al, 2003;Verma et al, 2012). Recently, CRISPR/Cas genome editing was shown in bovine iPSCs (Heo et al, 2014).…”

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

“…The pre-implantation embryo stage is generally not favoured for genome editing as it would most likely lead to a mosaic individual and possibly to more unforeseen and unforeseeable detrimental effects. Various studies in different animal models have demonstrated the feasibility of gene-editing in animals at the zygote stage (Yoshimi et al, 2014;Heo et al, 2015;Han et al, 2014, Kang et al, 2014. The potential of the technology to prevent the onset of a genetic disorder in mice was demonstrated by the studies of Wu et al (2013) and Long et al (2014), respectively for cataract and Duchenne muscular dystrophy.…”

Don’t edit the human germ line

“…Studies using gene-editing in animals such as rats 5 , cattle 6 , sheep 7 and pigs 8 , indicate that it is possible to delete or disable genes in an embryo -a simpler process than actually correcting DNA sequences -in only some of the cells.…”