Research Note: Generation of Ovalbumin-null Chickens and Characterization of Altered Protein Compositions in Their Egg Whites

Chickens are considered an efficient bioreactor platform for production of recombinant proteins due to their high egg laying rate and high capacity to produce proteins in egg white. Ovalbumin (OVAL) comprises 54% of egg white proteins, and targeted insertion of a recombinant protein construct into the OVAL locus leads to significant accumulation of the recombinant protein in egg white. However, it was reported that embryos could not develop or hatch from eggs laid by heterozygous OVAL -knockout hens in which OVAL gene was replaced by foreign protein coding sequences, a limitation that restricted the generation of homozygous OVAL -knockout chickens. In this study, we specifically targeted the OVAL locus using CRISPR/Cas9 and successfully generated OVAL -null chickens by mating heterozygous individuals. Both heterozygous and homozygous OVAL knockout embryos developed and hatched, notably, we found that embryos could develop and hatch from OVAL-deficient eggs, although the hatching rate was reduced by 52.30±14.42%. Furthermore, analysis revealed that concentrations of other major egg white proteins increased, indicating a compensatory accumulation of proteins in response to the removal of OVAL. Collectively, this study demonstrates that OVAL -null chicken lines can be established and that these chickens produce eggs with altered egg white protein compositions.