Innovative approaches to genome editing in avian species

Abstract

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal. In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells (PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing (STAGE). The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.

Keywords: Avian; CRISPR; Genome engineering; PGCs; Sperm; TALEN.

Fig. 1

Chicken, turkey, and quail sperm incubated with Lipofectamine® 2000 and BLOCK-iT™ fluorescently labelled RNA. The top panel shows unprocessed sperm, where poor transfection of the labelled RNA to the sperm is seen. The bottom panel shows STAGE processed sperm, where these is clearly increased transfection of the labelled RNA to the sperm. Quail pictures taken by Olivier Serralbo of the Monash Transgenic Quail Facility