Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene

Authors: JUNG, KYUNG MIN - University Of Missouri; KLEIN, RACHEL - University Of Missouri; MONY, SABRINA - University Of Missouri; Chen, Paula; LEE, KIHO - University Of Missouri; LEE, HONG JO - University Of Missouri

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2026
Publication Date: 2/6/2026
Citation: Jung, K., Klein, R., Mony, S., Chen, P.R., Lee, K., Lee, H. 2026. Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene. Poultry Science. 105(4). https://doi.org/10.1016/j.psj.2026.106585.
DOI: https://doi.org/10.1016/j.psj.2026.106585

Interpretive Summary: Genome editing technologies can be used to improve various production traits in poultry. However, these technologies have not been as extensively developed for avians compared to other species, such as mammals. The goal of this study was to improve upon genome editing in poultry cells, specifically the insertion of novel genes, to expand the current toolkit. This validation demonstrated that these genes could be inserted at precise locations and can serve as a basis for creation of genome-edited chickens using this method.

Technical Abstract: Achieving stable and efficient transgene expression is a key challenge in advancing avian genome engineering. Although viral vector-based and piggyBac-mediated transgenesis have been widely used in chickens, both approaches are prone to epigenetic silencing, leading to inconsistent, tissue-specific, and often diminished expression over time. This variability limits used of transgenes requiring robust and long-term expression across multiple tissues. In mammals, site-specific integration into genomic safe harbor loci, such as Rosa26, has enabled stable and predictable transgene expression without disrupting endogenous gene function; however, such strategy has not been established in birds. In this research, we hypothesized that integrating Cas9 into endogenous housekeeping genes (the ACTB and GAPDH) could achieve efficient gene editing in chickens through stable and ubiquitous transgene expression. Using two different approaches, 3'-targeted gene insertion and gene tagging, we inserted Cas9 and GFP cassettes into defined genomic loci in chicken DF-1 cells. Both approaches exhibited stable expression of transgenes in the cells, and functional assays confirmed that Cas9 showed highly efficient nuclease activity following guide RNA delivery. Additionally, we derived single-cell clones stably expressing Cas9, enabling uniform and reproducible genome editing in downstream applications. Targeted insertion of transgenes into active housekeeping genes as candidate safe harbor loci mitigates the limitations of random integration and promoter silencing, offering a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.