Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR era in the field of avian genome engineering.

Graphical Abstract

Gene knock-ins depending on the strategy of transgenesis can lead to either random or targeted integrations. Application of retrovirus (RV)-mediated transgenesis, lentivirus (LV) mediated transgenesis, transgenesis by random integration of plasmid (RIP), and transgenesis by transposons (TPs) leads to random integration of the transgene in the genome. In contrast, strategies for targeted and precise integration in the genome include homologous recombination (HR)-based transgenesis, recombinase-mediated transgenesis (RMT), TALEN-mediated transgenesis, and CRISPR-mediated transgenesis. Accordingly, the research on genome engineering in transgenic animals can be allocated to two periods of pre-CRISPR and the CRISPR era. The CRISPR/Cas9 technology has provided the most precise genome transgenesis outcomes in the engineering of the avian genome. The bottom half of the graph schematically depicts an overall outline for an ideal targeted knock-in using chicken primordial germ cells (cPGCs). cPGCs are isolated from embryonated eggs (Hamburger Hamilton; HH 13-15). The transgene is Knocked-in in the active (genome safe harbor; GSH) regions or the topologically associating domains (TADs) which will be de-repressed in the producing tissue. Genetically engineered cPGCs are then injected into the embryonated eggs (HH 13-15) from the dorsal aorta. These cells migrate in the blood circulation and home into the gonads of the 6-day-old embryo (HH 26-28). Genetically engineered roosters that included these cells in their gonads are reared and mated with wild-type hens. The reared adult genetically engineered hens in the next generation may have the transgene in the targeted locus of terminally differentiated cells such as the tubular gland cells of the magnum. In the tubular gland cells of the magnum, the recombinant biopharmaceutical can be secreted into the egg white if the transgene is knocked-in in the ovalbumin or a GSH locus active in these cells.