Establishing efficient multi-gene editing tools for papaya

Abstract Papaya is a major tropical fruit crop with notable nutritional and economic value, yet its genetic improvement through modern breeding technologies faces substantial challenges. The traditional tissue culture process is both labor-intensive and time-consuming, causing gene-editing advancements in papaya to lag behind those in other crops. To overcome these obstacles, we developed a tissue culture–independent hairy root system in papaya, which enables efficient gene editing and significantly enhances the application and development of editing tools. This innovative platform allows for the pre-assessment of editing efficiency and supports the establishment of adenine base editor (ABE) and cytosine base editor (CBE) tools in papaya, thereby mitigating the high failure costs associated with the lengthy cycle of conventional genetic transformation. Utilizing this system, we pre-tested sgRNA activity and achieved high editing efficiency of CpWIP3 during stable transformation. Additionally, through promoter screening, we successfully developed ABE and CBE tools, marking the first precise single-nucleotide editing system in papaya. This gene-editing system provides a crucial platform for advancing functional genomics and accelerating precision breeding in papaya.