PS4-8. Efficient DNA-free Genome Editing in Chicken Cells by Delivering mRNA and Protein Form of cas9.

Abstract Genome editing in avian systems has largely relied on plasmid-based CRISPR systems and antibiotic selection to achieve successful editing. However, plasmid delivery can result in prolonged nuclease expression and potential unintended DNA integration. In addition, selection-based enrichment may mask the intrinsic genome editing performance of CRISPR tools. Therefore, efficient DNA-free and selection-free genome editing strategies are needed. This study evaluated DNA-free CRISPR genome editing strategies in chicken cells using ribonucleoprotein (RNP) complexes and SpCas9 mRNA. Chicken fibroblast (DF-1) cells were nucleofected to target the germ cell-specific gene DAZL and the housekeeping gene ACTB. Editing outcomes were assessed by indel analysis under optimized conditions without antibiotic selection. RNP delivery and SpCas9 mRNA produced high editing efficiencies across all target loci, reaching up to 89% and 93% at the ACTB locus, respectively, whereas plasmid-based editing without selection peaked at 23% efficiency. These results demonstrate that efficient, selection-free genome editing can be achieved in chicken cells using DNA-free CRISPR delivery formats. This study provides a framework for safer and more reliable genome editing in avian systems and highlights possibility of producing footprint free genome-edited poultry using the approaches.