A fast-track system for transgene-free genome editing in oil palm (Elaeis guineensis Jacq.) via biolistic delivery of CRISPR/Cas9 ribonucleoprotein (CRISPR-RNP)

Abstract

The CRISPR/Cas9 system is a powerful tool for plant genome editing, but in oil palm, most platforms still rely on DNA-based delivery. Here, we report the first CRISPR/Cas9 Ribonucleoprotein (CRISPR-RNP)-mediated genome editing protocol for oil palm using biolistic transformation of embryogenic calli (EC). RNP complexes (RNP5/RNP6/RNP7, with or without 35SmDsRED2) targeting three sites in the phytoene desaturase (EgPDS) gene were successfully delivered. Targeted mutations were confirmed at three developmental stages: EC, polyembryoid-to-shoot (PES), and rooted plantlets (RPs). Edited RPs showed mutation frequencies of 24.4–29.1%, producing albino and variegated shoots with editing efficiencies of 10–84%. Importantly, this approach reduced regeneration time from EC to edited RPs by fourfold compared to conventional transgenic methods. This protocol provides the first demonstration of transgene-free, RNP-based editing in oil palm and offers a rapid, efficient platform for functional genomics and crop improvement.