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<p>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 <i>phytoene desaturase</i> (<i>EgPDS</i>) 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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年10月15日

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  • Jamaludin Norfaezah,
  • Mat Yunus Abdul Masani,
  • A. Rahman Nurfahisza,
  • Bohari Bahariah,
  • M. D. Piji Mohd Al-Akmarul Fizree,
  • Noor Azmi Shaharuddin,
  • Chai-Ling Ho,
  • Omar Abdul Rasid,
  • Ghulam Kadir Ahmad Parveez

摘要

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.