An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis)

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

Micro-RNA-Regulated SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Expression and Cytokinin Accumulation Distinguish Early-Developing Male and Female Inflorescences in Oil Palm (Elaeis guineensis)

“…For the moment, tools for the investigation of gene functions in oil palm are limited and constrained by the large size and long life cycle of this plant [ 56 ]. Nevertheless, progress has been made in the development of transient and stable genetic transformation methods, including CRISPR-based technology [ 57 ], while heterologous expression in more experimentally amenable species such as rice [ 58 ] may also offer solutions for functional validation studies.…”

Is CRISPR/Cas9 a way forward to fast-track genetic improvement in commercial palms? Prospects and limits

“…The electroporation method is used to identify the mutation rate and sgRNA efficiency ( Figure 4C ). The electroporation of Cas9 and sgRNA constructs resulted in a cleavage frequency of up to 25.49% in oil palm protoplasts ( Liu et al., 2016 ; Yeap et al., 2021 ). Using plant tissue culture to enhance and maintain genetic transformation has become a tool for scientists those involved in enhancement of agricultural production.…”

“…However, it is not well known whether the expression cassette and CRISPR/Cas9 vector successfully transformed into oil palm ( Aprilyanto et al., 2019 ). Above mentioned studies report that CRISPR/Cas9 successfully use in oil palm genome editing ( Yeap et al., 2021 ). In another study, the oil palm mesophyll protoplasts were extracted from unopened spear leaves ( Masani et al., 2013 ).…”

“…DNA insertions and deletions (InDels) in the EgBRI1 gene cause a decreased plant elongation phenotype. At the same time, leaf apex necrosis is developed from base substitution events in transgenic palms ( Yeap et al., 2021 ). In a recent study, the Nuzhdin Lab (USA), in partnership with the MPOB perform sequencing of more then 200 E. oleifera and E. guineensis palm trees from Southeast Asia, Africa, Central and South America (Gene bank accession PRJNA434010) ( Ithnin et al., 2021 ).…”

“…In the past few years, a concern was raised about GMOs related to foreign DNA, which could limit the widespread use of CRISPR technology ( Woo et al., 2015 ). CRISPR/Cas9 constructs are transferred into the plant via direct or indirect DNA delivery systems ( Masani et al., 2014 ; Budiani et al., 2018 ; Wolter and Puchta, 2018 ; Darmawan et al., 2020 ; Yeap et al., 2021 ). The CRISPR/Cas9 construct has a good chance of being incorporated into the plant’s genome throughout this process ( Zhang et al., 2016 ).…”