Genome edited sheep and cattle

Abstract

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

Fig. 1

The MSTN TALENs. a 359 bp of the bovine MSTN gene sequence showing the TALEN binding sites (red boxes) and the primers (green boxes) used to amplify the region for the surveyor nuclease assay. The base coloured blue dictates the position of the mismatch in the ovine sequence in which it is G rather than A. b The surveyor nuclease assay results for the TALEN transfected bovine and ovine fibroblasts. gDNA extracted from transfected cells was treated with and without nuclease. (Color figure online)

Fig. 2

MSTN edited animals. a The live born bull (bull #1: left) and heifer calf (right). b The readily observed phenotypic difference between bull #1 (right) and the wild-type heifer (left). c The edited lamb

Fig. 3

The MSTN editing events. An alignment of the bovine and ovine WT sequences and the alleles present in each of the edited animals. The TALEN binding sites are highlighted on the WT sequences, the ovine mismatch is underlined and the corresponding amino acid change is indicated on the right