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Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System (RTDS™) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

The European Commission requested the EFSA Panel on Genetically Modified Organisms (GMO) to assess whether section 4 (hazard identification) and the conclusions of EFSA's Scientific opinion on the risk assessment of plants developed using zinc finger nuclease type 3 technique (ZFN‐3) and other site‐directed nucleases (SDN) with similar function are valid for plants developed via SDN‐1, SDN‐2 and oligonucleotide‐directed mutagenesis (ODM). In delivering this Opinion, the GMO Panel compared the hazards associated with plants produced via SDN‐1, SDN‐2 and ODM with those associated with plants obtained via both SDN‐3 and conventional breeding. Unlike for SDN‐3 methods, the application of SDN‐1, SDN‐2 and ODM approaches aims to modify genomic sequences in a way which can result in plants not containing any transgene, intragene or cisgene. Consequently, the GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDN‐3 are not relevant to plants obtained via SDN‐1, SDN‐2 or ODM as defined in this Opinion. Overall, the GMO Panel did not identify new hazards specifically linked to the genomic modification produced via SDN‐1, SDN‐2 or ODM as compared with both SDN‐3 and conventional breeding. Furthermore, the GMO Panel considers that the existing Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are sufficient but are only partially applicable to plants generated via SDN‐1, SDN‐2 or ODM. Indeed, those guidance documents’ requirements that are linked to the presence of exogenous DNA are not relevant for the risk assessment of plants developed via SDN‐1, SDN‐2 or ODM approaches if the genome of the final product does not contain exogenous DNA. This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2020.EN-1972/full

The European Commission requested EFSA to provide an overview on the risk assessment of plants developed through new genomic techniques (NGTs), taking into account its previous scientific opinions, its ongoing work on the topic as well as opinions published by competent authorities and national institutions since 2012, where available. In this report, NGTs are defined as techniques capable to change the genetic material of an organism and have emerged or developed since the adoption of the 2001 genetically modified organism (GMO) legislation. EFSA considered 16 scientific opinions issued by European member states (‘MS opinions’) as well as three EFSA GMO Panel scientific opinions on NGTs. A procurement to evaluate and summarise the MS opinions was conducted. Relevant information on the description of each NGT and information on the risk assessment of plants developed through one or a combination of the defined NGTs was extracted and summarised. The baseline for the types and nature of NGTs to be included in this report was defined based on the JRC, 2011 report on new plant breeding techniques as well as on the Explanatory Note on New Techniques in Agricultural Biotechnology from the European Commissioner for Health and Food Safety (EC‐SAM, 2017) for some more recently developed NGTs, taking into account the NGT definition provided by the European Commission for this mandate. EFSA was not requested to develop new opinions on plants developed through specific NGTs, and thus, no critical appraisal of the reviewed scientific opinions was carried out. This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-1973/full

The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture.

Genome editing tools have the potential to change the genomic architecture of a genome at precise locations, with desired accuracy. These tools have been efficiently used for trait discovery and for the generation of plants with high crop yields and resistance to biotic and abiotic stresses. Due to complex genomic architecture, it is challenging to edit all of the genes/genomes using a particular genome editing tool. Therefore, to overcome this challenging task, several genome editing tools have been developed to facilitate efficient genome editing. Some of the major genome editing tools used to edit plant genomes are: Homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), pentatricopeptide repeat proteins (PPRs), the CRISPR/Cas9 system, RNA interference (RNAi), cisgenesis, and intragenesis. In addition, site-directed sequence editing and oligonucleotide-directed mutagenesis have the potential to edit the genome at the single-nucleotide level. Recently, adenine base editors (ABEs) have been developed to mutate A-T base pairs to G-C base pairs. ABEs use deoxyadeninedeaminase (TadA) with catalytically impaired Cas9 nickase to mutate A-T base pairs to G-C base pairs.

Background Plant breeding is a developing process and breeding methods have continuously evolved over time. In recent years, genome editing techniques such as clustered regularly interspaced short palindromic repeats/CRISPR associated proteins (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFN), meganucleases (MN) and oligonucleotide-directed mutagenesis (ODM) enabled a precise modification of DNA sequences in plants. Genome editing has already been applied in a wide range of plant species due to its simplicity, time saving and cost-effective application compared to earlier breeding techniques including classical mutagenesis. Although genome editing techniques induce much less unintended modifications in the genome (off-target effects) compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they might cause genomic instability, cytotoxicity and cell death. Methods The aim of this systematic map is to address the following primary question: “What is the available evidence for the application of genome editing as a new tool for plant trait modification and the potential occurrence of associated off - target effects” ? The primary question will be considered by two secondary questions: One is aimed at the traits being modified by genome editing in plants and the other explores the occurrence of off-target effects. The systematic map will focus on model plants as well as on plants produced for agricultural production that were subjected to genome editing techniques. Academic and grey literature will be searched in English and German language. Inclusion/exclusion criteria were developed for the two secondary questions and will be applied on title/abstract and full text stage. Included studies will be catalogued in a searchable and open access database and study results will be summarized using descriptive statistics. Furthermore, the extracted data will serve as a preparatory step for further in-depth analysis, e.g. by a systematic review.

Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

The Photosystem II (PSII) core antenna chlorophyll alpha-binding protein, CP47, contains six membrane-spanning a-helices separated by five hydrophilic loops: A-E. To identify important hydrophilic cytosolic regions, oligonucleotide-directed mutagenesis was employed to introduce short segment deletions into loops B and D, and the C-terminal domain. Four strains carrying deletions of between three and five residues were created in loop B. Two strains, with deletions adjacent to helices II and III, did not assemble PSII; however, the mutants Delta(F123-D125) and Delta(R127-S131) remained photoautotrophic with near wild-type levels of assembled reaction centers. In contrast, all deletions introduced into loop D, connecting helices IV and V, failed to assemble significant levels of PSII and were obligate photoheterotrophic mutants. However, deletions in the C-terminal domain did not prevent the assembly of PSII reaction centers although the mutant Delta(S471-T473), with a deletion adjacent to helix VI, exhibited retarded Q(A)(-) oxidation kinetics and the PSII-specific herbicide, atrazine, bound less tightly in the Delta(S471-T473) and Delta(F475-D477) strains. Deletions in the C-terminal domain also created mutants with large protein aggregates that were recognized by an antibody raised against the PSII reaction center D1 protein. Low-temperature fluorescence emission spectra of photoautotrophic strains carrying deletions in either the C-terminal domain or loop B did not provide evidence for impaired energy transfer from the phycobilisomes to the PSII reaction center. The data therefore suggest an important structural role for loop D in the assembly of PSII and a potential interaction between the C-terminal domain of CP47 and the PSII reaction center that, when perturbed, results in photoinduced protein aggregates involving the D1 protein.

Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-[14C]dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutants in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.

The Photosystem II (PSII) core antenna chlorophyll a-binding protein, CP47, contains six membrane-spanning α-helices separated by five hydrophilic loops: A-E. To identify important hydrophilic cytosolic regions, oligonucleotide-directed mutagenesis was employed to introduce short segment deletions into loops B and D, and the C-terminal domain. Four strains carrying deletions of between three and five residues were created in loop B. Two strains, with deletions adjacent to helices II and III, did not assemble PSII; however, the mutants Δ(F123-D125) and Δ(R127-S131) remained photoautotrophic with near wild-type levels of assembled reaction centers. In contrast, all deletions introduced into loop D, connecting helices IV and V, failed to assemble significant levels of PSII and were obligate photoheterotrophic mutants. However, deletions in the C-terminal domain did not prevent the assembly of PSII reaction centers although the mutant Δ(S471-T473), with a deletion adjacent to helix VI, exhibited retarded Q^sub A^^sup -^ oxidation kinetics and the PSII-specific herbicide, atrazine, bound less tightly in the Δ(S471-T473) and Δ(F475-D477) strains. Deletions in the C-terminal domain also created mutants with large protein aggregates that were recognized by an antibody raised against the PSII reaction center D1 protein. Low-temperature fluorescence emission spectra of photoautotrophic strains carrying deletions in either the C-terminal domain or loop B did not provide evidence for impaired energy transfer from the phycobilisomes to the PSII reaction center. The data therefore suggest an important structural role for loop D in the assembly of PSII and a potential interaction between the C-terminal domain of CP47 and the PSII reaction center that, when perturbed, results in photoinduced protein aggregates involving the D1 protein.[PUBLICATION ABSTRACT]