One-Step Genetic Modification by Embryonic Doral Aorta Injection of Adenoviral CRISPR/Cas9 Vector in Chicken.

In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63-11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16-4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species.