CRISPR/Cas9-based programmable genome editing in chickens: concepts, applications and regulatory issues.

The advent of genetics, molecular biology, and genome sequencing has rapidly accelerated the development of elite genetic lines across various species, including poultry. It is now possible to introduce intra- or inter-species single nucleotide polymorphisms into chicken lines to enhance productivity. This advancement may mark the beginning of a new agricultural revolution, dramatically reducing the time required to improve poultry lines for commercial production environments. Transgenic technologies, including lentiviral vectors and piggyBac transposition, have enabled the generation of animals expressing exogenous genes. The emergence of new genome-editing tools is transforming avian biotechnology, allowing the creation of customized organisms for specific traits. Genome editing has become the most efficient method for studying gene function. First and second generation tools, such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs), are limited by complex design and off-target effects. In contrast, the third generation Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9), represents a significant breakthrough. It encompasses guided RNA (gRNA) and the Cas9 endonuclease which together target specific DNA sequences and induces double-strand breaks that are repaired via error-prone non-homologous end joining, frequently causing insertions or deletions that disrupt gene function. Targeting specificity is achieved through gRNA-DNA base pairing and recognition of a protospacer adjacent motif by Cas9. Beyond gene knockout, CRISPR/Cas9 enables functional analysis of non-coding elements such as enhancers and insulators. Delivered via plasmid systems, Cas9 and gRNA are transiently expressed and degrade within 48-72 h, leaving no permanent genetic footprint. Since no exogenous DNA is integrated, this approach is generally considered less contentious than traditional transgenic methods in the context of genetically modified organism regulation. CRISPR/Cas9 has diverse applications in poultry, including enhancing disease resistance to avian influenza and Marek's disease, improving productivity traits such as growth, feed efficiency, and egg-laying, and enabling early in-ovo sexing to address ethical concerns around male chick culling. It also allows control of reproductive traits for breeding management, supports bio-pharming by producing therapeutic proteins or vaccines in eggs, and facilitates functional genomics through precise gene knockouts to study development, immunity, and metabolism.