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DNA-free CRISPR/Cas9 genome editing system for oil palm protoplasts using multiple ribonucleoproteins (RNPs) complexes


Norfaezah, Jamaludin and Masani, Mat Yunus Abdul and Fizree, MD Piji Mohd Al-Akmarul and Bahariah, Bohari and Shaharuddin, Noor Azmi and Ho, Chai Ling and Rasid, Omar Abdul and Parveez, Ghulam Kadir Ahmad (2024) DNA-free CRISPR/Cas9 genome editing system for oil palm protoplasts using multiple ribonucleoproteins (RNPs) complexes. Industrial Crops and Products, 208. art. no. 117795. pp. 1-12. ISSN 0926-6690


CRISPR/Cas9 is a powerful genome editing tool applicable to diverse plant species. However, the application of CRISPR/Cas9-ribonucleoproteins (RNPs) in oil palm (Elaeis guineensis) has not yet been reported. In this paper, the specificity, efficiency, and sensitivity of CRISPR/Cas9-RNPs in oil palm were evaluated by assessing the effect of different amounts of Cas9 and heat stress using E. guineensis phytoene desaturase (EgPDS) as the target gene. Seven gRNAs were designed and combined with Cas9 protein to form three combinations of RNPs complexes; RNP1/RNP2, RNP3/RNP4, and RNP5/RNP6/RNP7. Assessment of optimal amount of Cas9 and heat stress using RNP5/RNP6/RNP7 demonstrated that 40 µg of Cas9 and heat treatment at 39ºC increased the genome editing efficiency up to 95 in oil palm protoplasts. The use of both optimal conditions produced targeted mutations at frequencies of up to 63.6 in protoplasts transformed with RNP1/RNP2 and RNP3/RNP4 and up to 100 with RNP5/RNP6/RNP7 combinations. Multiple site mutagenesis at two to three target sites with fragment removal up to 179 bp, 69 bp, and 816 bp occurred at high frequencies producing editing efficiency and a Knockout Score (KO) of 100. Altogether, this study demonstrates a promising, highly efficient, and precise transgene-free CRISPR/Cas9-RNPs genome editing platform in oil palm that also holds enormous potential for application in other transformation-recalcitrant species. © 2023 Elsevier B.V.

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Additional Metadata

Item Type: Article
Divisions: Faculty of Biotechnology and Biomolecular Sciences
DOI Number: https://doi.org/10.1016/j.indcrop.2023.117795
Publisher: Elsevier
Keywords: CRISPR/Cas9; Genome editing; Oil palm; Phytoene desaturase; Ribonucleoproteins
Depositing User: Ms. Nuraida Ibrahim
Date Deposited: 08 Feb 2024 02:55
Last Modified: 08 Feb 2024 02:55
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1016/j.indcrop.2023.117795
URI: http://psasir.upm.edu.my/id/eprint/105666
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