scholarly journals The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods

2017 ◽  
Author(s):  
Jit Ern Chen ◽  
Adrian C. Barbrook ◽  
Guoxin Cui ◽  
Christopher J. Howe ◽  
Manuel Aranda
Keyword(s):  
Genome Editing ◽  
Nuclear Transformation ◽  
Great Success ◽  
Transformation Techniques ◽  
Chloramphenicol Resistance ◽  
Symbiodinium Microadriaticum ◽  
Silicon Carbide Whiskers ◽  
Atrazine Resistance ◽  
Broad Variety ◽  
Transformation Methods

AbstractModern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and silicon carbide whiskers. Chloroplast-targeted transformation were attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein that confers atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance to Symbiodinium microadriaticum strain CCMP2467.

scholarly journals In planta genome editing with CRISPR/Cas9 ribonucleoproteins

2021 ◽  
Author(s):  
Yuya Kumagai ◽  
Yuelin Liu ◽  
Haruyasu Hamada ◽  
Weifeng Luo ◽  
Jianghui Zhu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Particle Bombardment ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Wheat Variety ◽  
In Planta ◽  
Culm Length ◽  
Direct Delivery ◽  
Transformation Methods

In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

scholarly journals Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jake L. Weissman ◽  
Philip L. F. Johnson
Keyword(s):  
Genome Editing ◽  
Viral Infections ◽  
Adaptive Immune System ◽  
Gene Families ◽  
Great Success ◽  
Theory Approach ◽  
Viral Pathogens ◽  
Microbial Life ◽  
Associated Proteins ◽  
Short Palindromic Repeat

ABSTRACT A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording “memories” of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection. IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to “remember” past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples (“metagenomes”), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.

scholarly journals Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii

2020 ◽  
Vol 9 (10) ◽  
pp. 2840-2850
Author(s):  
Max Angstenberger ◽  
Francesco de Signori ◽  
Valeria Vecchi ◽  
Luca Dall’Osto ◽  
Roberto Bassi
Keyword(s):  
Homologous Recombination ◽  
Chlamydomonas Reinhardtii ◽  
Genome Editing ◽  
Nuclear Transformation ◽  
Cell Synchronization

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

2019 ◽  
Vol 20 (5) ◽  
pp. 1155 ◽  
Author(s):  
Guillaume Bernard ◽  
David Gagneul ◽  
Harmony Alves Dos Santos ◽  
Audrey Etienne ◽  
Jean-Louis Hilbert ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mutation Frequency ◽  
Function Analysis ◽  
Expression System ◽  
Crop Improvement ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
A Genome ◽  
Transformation Methods ◽  
Biallelic Mutations

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

scholarly journals Beyond Correlation: Acoustic Transformation Methods for the Experimental Study of Emotional Voice and Speech

2020 ◽  
pp. 175407392093454
Author(s):  
Pablo Arias ◽  
Laura Rachman ◽  
Marco Liuni ◽  
Jean-Julien Aucouturier
Keyword(s):  
Experimental Study ◽  
Signal Processing ◽  
Open Source ◽  
Real Time ◽  
Acoustic Analysis ◽  
Audio Signal ◽  
Audio Signal Processing ◽  
Transformation Techniques ◽  
Transformation Methods ◽  
Voice And Speech

While acoustic analysis methods have become a commodity in voice emotion research, experiments that attempt not only to describe but to computationally manipulate expressive cues in emotional voice and speech have remained relatively rare. We give here a nontechnical overview of voice-transformation techniques from the audio signal-processing community that we believe are ripe for adoption in this context. We provide sound examples of what they can achieve, examples of experimental questions for which they can be used, and links to open-source implementations. We point at a number of methodological properties of these algorithms, such as being specific, parametric, exhaustive, and real-time, and describe the new possibilities that these open for the experimental study of the emotional voice.

Identification of Nematicidal Fatty Acids and Triglycerides from Seeds of Jubaea chilensis by GC-EI-MS and Chemical Transformation Methods

2005 ◽  
Vol 60 (7-8) ◽  
pp. 527-533 ◽  
Author(s):  
Jian-Qiao Gu ◽  
C. Mark Eppler ◽  
Gloria Montenegro ◽  
Scott D. Timmins ◽  
Barbara N. Timmermann
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Chemical Transformation ◽  
Methyl Esters ◽  
Inhibitory Effects ◽  
Transformation Techniques ◽  
Bioassay Guided Fractionation ◽  
Transformation Methods ◽  
First Time

Nematicidal bioassay-guided fractionation of the n-hexane extract of the seeds of Jubaea chilensis led to the identification of eight known fatty acids and a mixture of triglycerides, reported for the first time for this species. In addition, their corresponding methyl esters were identified to be artifacts generated during the extraction and isolation procedures by using GC-EI-MS and chemical transformation methods. The fatty acid composition of the triglycerides was analyzed by GC-EI-MS and chemical transformation techniques. Among the 17 compounds, only lauric acid and myristic acid exhibited significant inhibitory effects on the movement of Caenorhabditis elegans with minimum inhibitory concentrations (MIC) of 75 μg/ml.

scholarly journals The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods

PLoS ONE ◽  
2019 ◽  
Vol 14 (2) ◽  
pp. e0211936 ◽  
Author(s):  
Jit Ern Chen ◽  
Adrian C. Barbrook ◽  
Guoxin Cui ◽  
Christopher J. Howe ◽  
Manuel Aranda
Keyword(s):  
Symbiodinium Microadriaticum ◽  
Transformation Methods
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