Improvement of chicken genome editing efficiency in vitro using ribonucleoprotein-mediated CRISPR/Cas9 delivery

BACKGROUND: Limitations in genome editing of avian species, including chicken, due to the inaccessibility of one-cell zygotes, have led to the manipulation of primordial germ cells (PGCs) as the primary approach for generating genetically engineered birds. Although plasmid-mediated delivery of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used, it has several limitations, including delayed nuclease activation, increased off-target effects, cytotoxicity, and the inability to apply in vivo selection strategies. Collectively, these limitations highlight the need to develop strategies that achieve high on-target activity at the initial editing step. RESULTS: In this study, we compared plasmid- and ribonucleoprotein (RNP)-mediated CRISPR/Cas9 delivery in Leghorn male hepatoma (LMH) cells and in PGCs targeting three loci: deleted in azoospermia like (DAZL), chicken vasa homologue (CVH), and stimulated by retinoic acid 8 (STRA8). RNP delivery showed comparable or higher cell viability and editing efficiency than plasmid delivery in both cell types. Insertion/deletion (indel) profiling revealed broader and more diverse mutation patterns with RNPs, consistent with a shift in repair pathway engagement toward microhomology-mediated end joining (MMEJ), which favors larger deletions. Off-target analysis further showed substantially reduced off-target editing and high specificity with RNP delivery. CONCLUSIONS: Together, these findings demonstrate that RNP delivery improves efficiency, reduces cytotoxicity, and improves precision, providing a more reliable platform for chicken genetic engineering.