scholarly journals Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Isidre Hooghvorst ◽  
Camilo López-Cristoffanini ◽  
Salvador Nogués
Keyword(s):  
Genome Editing ◽  
Cucumis Melo ◽  
Phytoene Desaturase ◽  
Desaturase Gene ◽  
Transformed Plants ◽  
Albino Plants ◽  
Cotyledonary Explants ◽  
Cas9 Protein ◽  
Albino Phenotype ◽  
Set Up

AbstractCRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42–45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

scholarly journals In planta test system for targeted cellular mutagenesis by injection of oligonucleotides to apical meristem of maize seedlings

2021 ◽  
Vol 43 (5) ◽  
Author(s):  
Feríz Rádi ◽  
Bettina Nagy ◽  
Györgyi Ferenc ◽  
Katalin Török ◽  
István Nagy ◽  
...  
Keyword(s):  
Genome Editing ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Stop Codon ◽  
Test System ◽  
Phytoene Desaturase ◽  
Vascular Bundles ◽  
Maize Seedlings ◽  
In Planta ◽  
Desaturase Gene

AbstractGenome-editing tools from Oligonucleotide-Directed Mutagenesis (ODM) to CRISPR system use synthetic oligonucleotides for targeted exchange of nucleotides. Presently, majority of genome-editing protocols are dependent on the in vitro cell or tissue culture systems with somaclonal variation, and limitations in plant regeneration. Therefore, here, we report an alternative in planta cellular test system for optimization of the ODM, based on the injection of oligonucleotide solution into the apical meristematic region of haploid maize seedlings. Using 5′-fluorescein-labeled oligonucleotides, we detected accumulation of synthetic DNA molecules in cells of the shoot apical meristem and of the vascular bundles of leaf primordia. For silencing or knocking down of the phytoene desaturase gene in somatic cells, 41-mer long single-stranded oligonucleotides with TAG stop codon were injected into maize seedlings. We detected out-growing M1 plantlets that developed leaves with white stripes or pale-green color. Confocal microscopy of white stripes showed that in addition to the chlorophyll fluorescence-deficient tissue region, chlorophyll containing cells are present in white stripes. The Ion Torrent sequencing of DNA samples from the white stripes indicated 0.13–1.50% read frequency for the TAG stop codon in the phytoene desaturase gene. Appearance of chlorotic abnormalities supports the mutagenic nature of oligonucleotide molecules after injection into the shoot apical meristem region of maize seedling. The described protocol provides basis for early seedling stage characterization of functionality of a mutagenic oligonucleotide with different chemistry and testing efficiency of various treatment combinations at plant level.

GENERATION OF TOBACCO PLANTS WITH EDITED GENOME: EVALUATION OF THE INFLUENCE OF CANCELLED ACTIVITY OF THE PHYTHOENE DESATURASE GENE ON PLANT DEVELOPMENT

2020 ◽  
pp. 346-349
Author(s):  
A. Kamionskaya ◽  
E. Kochieva ◽  
M. Eldarov ◽  
A. Shchennikova ◽  
M. Slugina
Keyword(s):  
Plant Development ◽  
Phytoene Desaturase ◽  
Flower Number ◽  
Desaturase Gene ◽  
Tobacco Plants ◽  
Albino Phenotype ◽  
Delayed Growth ◽  
Carotenoid Pathway

Tobacco plants with the edited genome were generated. The synthesis of phytoene desaturase, one of the main enzymes of the carotenoid pathway, is partially impaired. Edited lines are characterized by mosaic albino phenotype, delayed growth, and significant reduction in the flower number in inflorescence.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

Silenced Phytoene desaturase Gene as a Scorable Marker for Plant Genetic Transformation

2014 ◽  
Vol 13 (2) ◽  
pp. 80-84
Author(s):  
Vikas Yadav Patade ◽  
Deepti Khatri ◽  
Atul Grover ◽  
Maya Kumari ◽  
Sanjay Mohan Gupta ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Phytoene Desaturase ◽  
Desaturase Gene

scholarly journals Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1311
Author(s):  
Liuru Su ◽  
Chenggang Shi ◽  
Xin Huang ◽  
Yiquan Wang ◽  
Guang Li
Keyword(s):  
Animal Model ◽  
Thermal Treatment ◽  
Genome Editing ◽  
Phylogenetic Position ◽  
Transcription Activator ◽  
Cell Stage ◽  
System A ◽  
Cas9 Nuclease ◽  
Cas9 Protein ◽  
Amphioxus Genome

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

scholarly journals Establishing CRISPR/Cas9 in Lipomyces starkeyi

2019 ◽  
Vol 2 (2) ◽  
pp. 51-52
Author(s):  
Zoe Lau ◽  
David Stuart ◽  
Bonnie Mcneil
Keyword(s):  
Protein Expression ◽  
Genome Editing ◽  
Oleaginous Yeast ◽  
Lipomyces Starkeyi ◽  
Guide Rna ◽  
Intracellular Lipids ◽  
Dry Cell Weight ◽  
Cas9 Protein ◽  
High Concentrations ◽  
Dry Cell

The goal of this project was to adapt the Yarrowia lipolytica plasmid based CRISPR/Cas9 system for usage in Lipomyces starkeyi. Lipomyces starkeyi is an oleaginous yeast, which synthesizes and stores high amounts of intracellular lipids. This specific yeast can store lipids at concentrations higher than 60% of its dry cell weight. Due to these high concentrations of lipids, L. starkeyi is a desired organism for the production of biofuels and other oleochemicals. However, there is a lack of knowledge and of genetic tools when trying to engineer the cells to produce these lipids for our use. The genome editing tool, CRISPR/Cas9 is efficient and simple, therefore desirable for the engineering of L. starkeyi. The goal was achieved by replacing the Y. lipolytica promoter with a L. starkeyi promoter, inserting guide RNA, as well as confirming cas9 protein expression.

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