Evaluation of transient DsRED gene expression in oil palm embryogenic calli

Fizree, Piji Mohd Al Akmarul; Shaharuddin, Noor Azmi; Ho, Chai Ling; Masura, Subhi Siti; Manaf, Mohamad Arif Abdul; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul

doi:10.1016/j.scienta.2019.108679

Cited by 4 publications

(1 citation statement)

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“…RFP, a red fluorescent protein, was used as a visual marker to gauge the efficiency of biolistic-mediated transformation. Oil palm embryogenic calli were co-bombarded with pAMDsRED [ 35 ], a plasmid-carrying gene encoding DsRED . The red fluorescent signals from the expression of the DsRED gene were readily detected at 48 h after the transformation, which confirmed the successful delivery of CRISPR/Cas9 vectors into oil palm calli (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

Bahariah

Masani

Fizree

et al. 2023

Journal of Genetic Engineering and Biotechnology

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Background CRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. Results A total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. Conclusion This technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

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Section: Resultsmentioning

confidence: 99%