scholarly journals In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

2021 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
In Planta ◽  
Crop Species ◽  
Increase Copy Number ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

AbstractAdvances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

scholarly journals In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

2021 ◽  
Vol 3 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
Wheat Dwarf Virus ◽  
In Planta ◽  
Crop Species ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

scholarly journals Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting

2018 ◽  
Author(s):  
Florian Hahn ◽  
Marion Eisenhut ◽  
Otho Mantegazza ◽  
Andreas P.M. Weber
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Strand Break ◽  
Double Strand Break ◽  
In Planta ◽  
End Joining ◽  
Trichome Formation ◽  
Break Site ◽  
Repair Template ◽  
Non Homologous End Joining

ABSTRACTThe CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination. While the first repair mechanism results in random mutations at the double-strand break site, homologous recombination uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by homologous recombination are very low. In this study, we compared two methods that have been reported to enhance frequencies of homologous recombination in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of homologous recombination events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by homologous recombination at frequencies of 0.12% using the in planta gene targeting approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

Characterization and applications of Type I CRISPR-Cas systems

2020 ◽  
Vol 48 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Claudio Hidalgo-Cantabrana ◽  
Rodolphe Barrangou
Keyword(s):  
Transcriptional Regulation ◽  
Genome Editing ◽  
Adaptive Immune System ◽  
Type I ◽  
Research Areas ◽  
Wide Range ◽  
Class 1 ◽  
Compact Class ◽  
Molecular Machinery ◽  
Repair Template

CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.

Artificial microRNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa)

Botany ◽  
2013 ◽  
Vol 91 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Julian C. Verdonk ◽  
Michael L. Sullivan
Keyword(s):  
Gene Silencing ◽  
Medicago Sativa ◽  
Target Gene ◽  
Target Genes ◽  
Mirna Precursor ◽  
Crop Species ◽  
Forage Crop ◽  
Ribonucleic Acids ◽  
Endogenous Gene ◽  
Medicago Sativa L

Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used, however they all have in common the artificial generation of single stranded small ribonucleic acids (RNAs) that are utilized by the endogenous gene silencing machinery of the organism. Artificial microRNAs (amiRNA) can be used to very specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana (L.) Heynh. and rice using endogenous microRNA (miRNA) precursors and has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrate that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.

scholarly journals Silicon Effects on the Root System of Diverse Crop Species Using Root Phenotyping Technology

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 885
Author(s):  
Pooja Tripathi ◽  
Sangita Subedi ◽  
Abdul Latif Khan ◽  
Yong-Suk Chung ◽  
Yoonha Kim
Keyword(s):  
Root System ◽  
Root Morphology ◽  
Morphological Traits ◽  
Crop Production ◽  
Spatial Configuration ◽  
Global Climate ◽  
Learning Technologies ◽  
Climate Change Scenarios ◽  
Crop Species ◽  
Wide Range

Roots play an essential function in the plant life cycle, as they utilize water and essential nutrients to promote growth and plant productivity. In particular, root morphology characteristics (such as length, diameter, hairs, and lateral growth) and the architecture of the root system (spatial configuration in soil, shape, and structure) are the key elements that ensure growth and a fine-tuned response to stressful conditions. Silicon (Si) is a ubiquitous element in soil, and it can affect a wide range of physiological processes occurring in the rhizosphere of various crop species. Studies have shown that Si significantly and positively enhances root morphological traits, including root length in rice, soybean, barley, sorghum, mustard, alfalfa, ginseng, and wheat. The analysis of these morphological traits using conventional methods is particularly challenging. Currently, image analysis methods based on advanced machine learning technologies allowed researchers to screen numerous samples at the same time considering multiple features, and to investigate root functions after the application of Si. These methods include root scanning, endoscopy, two-dimensional, and three-dimensional imaging, which can measure Si uptake, translocation and root morphological traits. Small variations in root morphology and architecture can reveal different positive impacts of Si on the root system of crops, with or without exposure to stressful environmental conditions. This review comprehensively illustrates the influences of Si on root morphology and root architecture in various crop species. Furthermore, it includes recommendations in regard to advanced methods and strategies to be employed to maintain sustainable plant growth rates and crop production in the currently predicted global climate change scenarios.

scholarly journals Citizen views on genome editing: effects of species and purpose

2021 ◽  
Author(s):  
Gesa Busch ◽  
Erin Ryan ◽  
Marina A. G. von Keyserlingk ◽  
Daniel M. Weary
Keyword(s):  
Disease Resistance ◽  
Product Quality ◽  
Genome Editing ◽  
Total Sample ◽  
Moral Foundations ◽  
Perceived Risks ◽  
Public Response ◽  
The Public ◽  
Wide Range ◽  
The Us

AbstractPublic opinion can affect the adoption of genome editing technologies. In food production, genome editing can be applied to a wide range of applications, in different species and with different purposes. This study analyzed how the public responds to five different applications of genome editing, varying the species involved and the proposed purpose of the modification. Three of the applications described the introduction of disease resistance within different species (human, plant, animal), and two targeted product quality and quantity in cattle. Online surveys in Canada, the US, Austria, Germany and Italy were carried out with a total sample size of 3698 participants. Using a between-subject design, participants were confronted with one of the five applications and asked to decide whether they considered it right or wrong. Perceived risks, benefits, and the perception of the technology as tampering with nature were surveyed and were complemented with socio-demographics and a measure of the participants’ moral foundations. In all countries, participants evaluated the application of disease resistance in humans as most right to do, followed by disease resistance in plants, and then in animals, and considered changes in product quality and quantity in cattle as least right to do. However, US and Italian participants were generally more positive toward all scenarios, and German and Austrian participants more negative. Cluster analyses identified four groups of participants: ‘strong supporters’ who saw only benefits and little risks, ‘slight supporters’ who perceived risks and valued benefits, ‘neutrals’ who showed no pronounced opinion, and ‘opponents’ who perceived higher risks and lower benefits. This research contributes to understanding public response to applications of genome editing, revealing differences that can help guide decisions related to adoption of these technologies.

scholarly journals Chromosomal Locus That Affects Pathogenicity of Rhodococcus fascians

2002 ◽  
Vol 184 (4) ◽  
pp. 1112-1120 ◽  
Author(s):  
Danny Vereecke ◽  
Karen Cornelis ◽  
Wim Temmerman ◽  
Mondher Jaziri ◽  
Marc Van Montagu ◽  
...  
Keyword(s):  
Krebs Cycle ◽  
Malate Synthase ◽  
Habitat Modification ◽  
Signal Molecules ◽  
In Planta ◽  
Chromosomal Locus ◽  
Physiological Alterations ◽  
Rhodococcus Fascians ◽  
Wide Range ◽  
Symptomatic Tissue

ABSTRACT The gram-positive plant pathogen Rhodococcus fascians provokes leafy gall formation on a wide range of plants through secretion of signal molecules that interfere with the hormone balance of the host. Crucial virulence genes are located on a linear plasmid, and their expression is tightly controlled. A mutant with a mutation in a chromosomal locus that affected virulence was isolated. The mutation was located in gene vicA, which encodes a malate synthase and is functional in the glyoxylate shunt of the Krebs cycle. VicA is required for efficient in planta growth in symptomatic, but not in normal, plant tissue, indicating that the metabolic requirement of the bacteria or the nutritional environment in plants or both change during the interaction. We propose that induced hyperplasia on plants represents specific niches for the causative organisms as a result of physiological alterations in the symptomatic tissue. Hence, such interaction could be referred to as metabolic habitat modification.

Short DNA sequences inserted for gene targeting can accidentally interfere with off‐target gene expression

2010 ◽  
Vol 24 (6) ◽  
pp. 1714-1724 ◽  
Author(s):  
Ingo D. Meier ◽  
Christian Bernreuther ◽  
Thomas Tilling ◽  
John Neidhardt ◽  
Yong Wee Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Targeting ◽  
Dna Sequences ◽  
Target Gene ◽  
Target Gene Expression

scholarly journals Constraint-based metabolic control analysis for rational strain engineering

2020 ◽  
Author(s):  
Sophia Tsouka ◽  
Meric Ataman ◽  
Tuure Hameri ◽  
Ljubisa Miskovic ◽  
Vassily Hatzimanikatis
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Metabolic Control ◽  
Metabolic Flux ◽  
Strain Engineering ◽  
Metabolic Control Analysis ◽  
Physiological Data ◽  
Response Analysis ◽  
Control Analysis ◽  
Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

scholarly journals Detectability of Pseudomonassyringae pv. aesculi from European Horse Chestnut Using Quantitative PCR Compared with Traditional Isolation

Forests ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1062
Author(s):  
Salome Schneider ◽  
Christopher Schefer ◽  
Joana Beatrice Meyer
Keyword(s):  
Quantitative Pcr ◽  
Bacterial Growth ◽  
Target Gene ◽  
Horse Chestnut ◽  
Inoculation Experiment ◽  
Copy Numbers ◽  
Infection Stage ◽  
Time Point

Bleeding cankers on horse chestnut trees (Aesculushippocastanum and Aesculus × carnea), caused by Pseudomonassyringae pv. aesculi, have been reported across Europe. In the present study, we show the successful detection of P. syringae pv. aesculi on symptomatic horse chestnut trees in Switzerland using quantitative PCR (qPCR). However, P. syringae pv. aesculi was also detected by qPCR on trees from which no isolate was obtained through cultivation. Reduced isolation success and low copy numbers of the target gene were correlated with the increasing age of symptomatic horse chestnut trees. The potential of detecting non-viable P. syringae pv. aesculi by qPCR was evaluated using an inoculation experiment with dead bacteria and detection by qPCR and cultivation. The detectability of DNA from P. syringae pv. aesculi cells dropped by 34.5% one day after inoculation and then decreased only slightly until the end of the experiment (22 days after inoculation). In contrast, no bacterial growth was observed at any time point after the inactivation of the bacteria. To protect horse chestnut trees, evaluating the viability and actual infection stage of the bacterium may play an important role.

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