Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Significance Current methods for CRISPR/Cas9-mediated gene knockout in avian species require isolation, culture, and genome modification of primordial germ cells in vitro for the subsequent injection into the extraembryonic blood vessel of the egg. These processes require highly skilled techniques and the establishment of optimized conditions. In the present study, the adenoviral CRISPR/Cas9 vector, optimized for expression of gRNA and the Cas9 gene in avian cells, was injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin gene, resulting in gray plumage of homozygous mutant quail. Implementation of this simplified avian knockout process can accelerate avian knockout studies involved in diverse areas of research and lead to the advancement of future agricultural applications.