Gene targeting, genome editing: from Dolly to editors

Abstract

One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these 'molecular scissors' have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way-but the most exciting period is just starting.

Keywords: CRISPR/Cas9; Cytoplasmic injection; Gene targeting; Genome editing; Livestock; SCNT or cloning; TALENs.

Fig. 1

Routes to genome edited livestock. Designer nucleases have been successfully used to modify both zygotes and somatic cells. Modification and selection of fibroblasts coupled with SCNT has resulted in the generation of HDR and NHEJ edited livestock. NHEJ edited animals have been produced via zygote CPI whereas, to date, HDR edited animals have not been reported from edited zygotes

Fig. 2

The utility of double strand breaks generated by genome editors. A cartoon depiction of the double strand break (DSB) repair mechanisms. Non homologous end joining (NHEJ) is an error prone process that re-joins the end of the DSB, often resulting in small insertions/deletions (blue) and subsequent gene disruption. Homology dependent repair is a faithful process that uses a homologous template to repair the DSB. Providing a repair template, either as a single stranded oligonucleotide or double stranded DNA, allows specific modifications (green) to be introduced to the genome. Creation of simultaneous DSBs flanking a region of the genome can result in deletion of the intervening sequence (yellow) and repair of the DSBs by either NHEJ or HDR. (Color figure online)

Fig. 3

A Timeline of genome edited livestock over the past 5 years highlighting specific milestones

Fig. 4

Live genome edited pigs produced by TALEN injection into zygotes. a Founder NHEJ animals born 2012 (Lillico et al. 2013). b Third generation piglets derived from NHEJ founder animals