Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.

Recently developed myostatin (MSTN) mutant quail and chickens demonstrated similar effects of MSTN on muscle and fat developments between avian and mammalian species. However, the effect of MSTN mutation on the quality of eggshells, an important avian specific characteristic, has not yet been investigated although egg production traits of mutant quail have been studied. In this study, several parameters for eggshell quality, including eggshell size, eggshell weight, eggshell breaking strength (EBS), and eggshell thickness, were all compared between MSTN mutant and wild-type (WT) eggs. MSTN mutant eggs had greater height and width along with heavier eggshell weight compared to WT eggs, which shows proportional improvement in egg size as affected by the MSTN mutation. However, EBS and eggshell thickness were decreased in mutant eggs compared to WT eggs. In addition, the palisade layer, the thickest and most important layer for the strength of an eggshell, was also decreased without a change in the number of vesicular holes. These data indicated that decreases in the thickness of the eggshell and the palisade layer would be a main factor contributing to a lower EBS in mutant eggs. MSTN mutant quail provide a useful model to better understand the function of MSTN on avian uterine cell development and eggshell biomineralization.