CRISPR/Cas9 Genome Editing in Livestock: Mechanisms, Delivery, Applications, and Regulatory Considerations

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein 9 (Cas9) system has emerged as the most transformative genome- editing technology in the history of animal biotechnology. Since the demonstration of its programmable nuclease activity in mammalian cells in early 2013, CRISPR/Cas9 has been rapidly adopted in livestock research, enabling modifications in cattle, pigs, sheep, goats, and poultry with a speed and precision that was inconceivable with earlier zinc- finger nuclease (ZFN) or transcription activator- like effector nuclease (TALEN) platforms. This review provides a comprehensive examination of the CRISPR/Cas9 mechanism, including guide RNA design principles, PAM sequence requirements, double- strand break repair pathways (NHEJ and HDR), and the growing family of Cas9 orthologs and variants. Delivery methods applicable to livestock zygotes and somatic cells — including microinjection, electroporation, lentiviral vectors, lipofection, and AAV — are systematically compared with respect to efficiency, off- target activity, and practical feasibility. A comprehensive survey of CRISPR/Cas9 applications in livestock covering disease resistance (PRRS, African swine fever), production trait improvement (myostatin knockout, cashmere enhancement, poledness), biomedical modelling, and xenotransplantation is provided. Methods for detecting and quantifying off- target editing events are discussed, along with the implications of off- target activity for food safety. The review concludes with a comparative analysis of regulatory frameworks across major jurisdictions, highlighting the spectrum from the permissive (Argentina) to the precautionary (European Union), and discussing the science- policy interface challenges that will determine the pace at which CRISPR- edited livestock products enter commercial production and consumer markets.