Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L)

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1-1 mannose without sucrose. Transgenic plants were verified using PCR analysis

Biolistic transformation of oil palm using the phosphomannose isomerase (pmi) gene as the positive selectable marker / Bahariah Bohari

The selectable marker system based on the Escherichia coli phosphomannose isomerase (pmi) gene was adapted for genetic transformation of oil palm. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate and uses mannose as the selection agent. This is to anticipate the future requirement of using non-antibiotic resistance genes for the commercialization of transgenic oil palm. The use of antibiotic or herbicide-based selection systems has caused much public concern due to inadequate knowledge of the agents’ impact on the environment and on human health. In this study, four transformation vectors, namely pMI3, pMI11, pMI3G and pMI11G were constructed for transforming oil palm. The pmi gene is driven by CaMV35S promoter in pMI3 and pMI3G, and Ubi1 promoter in pMI11 and pMI11G. gusA gene was also included in the pMI3G and pMI11G constructs. The gene constructs were transferred into oil palm embryogenic calli via biolistic-mediated transformation. Bombarded calli were selected on medium supplemented with mannose as a carbon source. Results from kill curve experiment indicated that oil palm embryogenic callus have little or no PMI activity and cannot utilize mannose as a carbon source. However, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize mannose efficiently as carbon source. For early identification of transgenic events, histochemical staining with 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Gluc) was used. Transgenic plants were confirmed by PCR and the transgene expression was detected using RT-PCR analysis. Transient expression results demonstrate that the Ubi1 promoter is more efficient than the CaMV35S promoter in oil palm embryogenic calli. The insertion verification was confirmed with 98% homology observed to its corresponding pmi gene from E. coli (Genbank accession no: M15380) via PCR direct sequencing. In conclusion, the results of this study indicated that mannose selection system can be used for oil palm transformation. Potentially this will make transgenic oil palm acceptable in the future

Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm

Abstract Background Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency. Results Here, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system’s efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs. Conclusion The results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm

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

Abstract 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

Designing gRNAs targeting oil palm phytoene desaturase (EgPDS) gene and construction of vectors for oil palm CRISPR/Cas9 study

The CRISPR/Cas9 system is a precise and versatile genetic tool used to induce site-specific manipulation in the targeted gene. This study elaborates on the strategy to select the gRNAs targeting the oil palm phytoene desaturase (EgPDS) gene, which complements CRISPR-GE and sequence analysis. Sixty-one gRNAs were selected from 167 gRNA candidates generated by the CRISPR-GE tool. These gRNA candidates were further analysed to meet the specific criteria, including the guanine (G) and cytosine (C) content of 35%-60% and the presence of preferable gRNA features. From 61 gRNAs, seven gRNAs were selected, which represent three different parts of the EgPDS gene in the genome. The efficiency of each gRNA candidate to cause cleavage on the target gene was confirmed by in vitro cleavage analysis. All seven gRNA candidates were then cloned into the pYLsgRNA-OsU6 cassette before being incorporated seamlessly into the final transformation vector, pYLCRISPR/Cas9P35S-H, via Gibson assembly. Three CRISPR/Cas9 transformation vectors targeting the EgPDS gene, namely pYLEgPDS1-35S-H, pYLEgPDS2-35S-H and pYLEgPDS3-35S-H, were successfully constructed. These constructs will be transformed into oil palm protoplasts via polyethylene glycol (PEG)- mediated transformation and subsequently into oil palm embryogenic calli via biolistic and Agrobacteriummediated transformation to determine the efficacy of the multiplex CRISPR/Cas9 system in oil palm genome

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

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