Relevant bibliographies by topics / Plant genome editing

Academic literature on the topic 'Plant genome editing'

Author: Grafiati
Published: 10 December 2022
Last updated: 28 January 2023

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Journal articles on the topic "Plant genome editing"

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Langner, Thorsten, Sophien Kamoun, and Khaoula Belhaj. "CRISPR Crops: Plant Genome Editing Toward Disease Resistance." Annual Review of Phytopathology 56, no. 1 (August 25, 2018): 479–512. http://dx.doi.org/10.1146/annurev-phyto-080417-050158.

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Genome editing by sequence-specific nucleases (SSNs) has revolutionized biology by enabling targeted modifications of genomes. Although routine plant genome editing emerged only a few years ago, we are already witnessing the first applications to improve disease resistance. In particular, CRISPR-Cas9 has democratized the use of genome editing in plants thanks to the ease and robustness of this method. Here, we review the recent developments in plant genome editing and its application to enhancing disease resistance against plant pathogens. In the future, bioedited disease resistant crops will become a standard tool in plant breeding.
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Li, Yizhen, Jing Liang, Bufang Deng, Yingli Jiang, Jingyan Zhu, Like Chen, Min Li, and Juan Li. "Applications and Prospects of CRISPR/Cas9-Mediated Base Editing in Plant Breeding." Current Issues in Molecular Biology 45, no. 2 (January 19, 2023): 918–35. http://dx.doi.org/10.3390/cimb45020059.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 system (Cas9) has been used at length to optimize multiple aspects of germplasm resources. However, large-scale genomic research has indicated that novel variations in crop plants are attributed to single-nucleotide polymorphisms (SNPs). Therefore, substituting single bases into a plant genome may produce desirable traits. Gene editing by CRISPR/Cas9 techniques frequently results in insertions–deletions (indels). Base editing allows precise single-nucleotide changes in the genome in the absence of double-strand breaks (DSBs) and donor repair templates (DRTs). Therefore, BEs have provided a new way of thinking about genome editing, and base editing techniques are currently being utilized to edit the genomes of many different organisms. As traditional breeding techniques and modern molecular breeding technologies complement each other, various genome editing technologies have emerged. How to realize the greater potential of BE applications is the question we need to consider. Here, we explain various base editings such as CBEs, ABEs, and CGBEs. In addition, the latest applications of base editing technologies in agriculture are summarized, including crop yield, quality, disease, and herbicide resistance. Finally, the challenges and future prospects of base editing technologies are presented. The aim is to provide a comprehensive overview of the application of BE in crop breeding to further improve BE and make the most of its value.
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Oh, Youngbin, Hyeonjin Kim, and Sang-Gyu Kim. "Virus-induced plant genome editing." Current Opinion in Plant Biology 60 (April 2021): 101992. http://dx.doi.org/10.1016/j.pbi.2020.101992.

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Huang, Yong, Meiqi Shang, Tingting Liu, and Kejian Wang. "High-throughput methods for genome editing: the more the better." Plant Physiology 188, no. 4 (February 3, 2022): 1731–45. http://dx.doi.org/10.1093/plphys/kiac017.

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Abstract During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies.
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Ricroch, Agnes E., Klaus Ammann, and Marcel Kuntz. "Editing EU legislation to fit plant genome editing." EMBO reports 17, no. 10 (September 14, 2016): 1365–69. http://dx.doi.org/10.15252/embr.201643099.

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Zhang, Chao, Shanhe Liu, Xuan Li, Ruixuan Zhang, and Jun Li. "Virus-Induced Gene Editing and Its Applications in Plants." International Journal of Molecular Sciences 23, no. 18 (September 6, 2022): 10202. http://dx.doi.org/10.3390/ijms231810202.

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CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.
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Memon, Abdulrezzak. "CRISPR/Cas9 Mediated Genome Editing in Crop Plants." Turkish Journal of Agriculture - Food Science and Technology 9, sp (January 5, 2022): 2396–400. http://dx.doi.org/10.24925/turjaf.v9isp.2396-2400.4810.

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Recently, most genomic research has focused on genome editing methods to develop new technologies that could be easy, reliable, and feasible to edit plant genomes for highly productive agriculture. Genome editing is based on alternating a specific target DNA sequence by adding, replacing, and removing DNA bases. This newest technology called CRISPR/Cas9 seems to be less time-consuming, more effective and used in many research areas of plant genetic research. CRISPR/Cas9 systems have many advantages in comparison with ZFNs and TALENs and has been extensively used for genome editing to many crop plant species. Around 20 crop species are successfully worked out for trait improvements, for example, yield improvement, disease resistance, herbicide tolerance, and biotic and abiotic stress management. This review paper will overview recent advances in CRISPR/Cas genome editing research in detail. The main focus will be on the use of CRISPR/Cas9 technology in plant genome research.
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Ezura, Hiroshi, and Kenji Miura. "Genome editing technologies for plant physiology." Plant Physiology and Biochemistry 131 (October 2018): 1. http://dx.doi.org/10.1016/j.plaphy.2018.07.007.

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Hua, Kai, Peijin Han, and Jian-Kang Zhu. "Improvement of base editors and prime editors advances precision genome engineering in plants." Plant Physiology 188, no. 4 (December 28, 2021): 1795–810. http://dx.doi.org/10.1093/plphys/kiab591.

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Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-mediated gene disruption has revolutionized biomedical research as well as plant and animal breeding. However, most disease-causing mutations and agronomically important genetic variations are single base polymorphisms (single-nucleotide polymorphisms) that require precision genome editing tools for correction of the sequences. Although homology-directed repair of double-stranded breaks (DSBs) can introduce precise changes, such repairs are inefficient in differentiated animal and plant cells. Base editing and prime editing are two recently developed genome engineering approaches that can efficiently introduce precise edits into target sites without requirement of DSB formation or donor DNA templates. They have been applied in several plant species with promising results. Here, we review the extensive literature on improving the efficiency, target scope, and specificity of base editors and prime editors in plants. We also highlight recent progress on base editing in plant organellar genomes and discuss how these precision genome editing tools are advancing basic plant research and crop breeding.
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Chen, Kunling, Yanpeng Wang, Rui Zhang, Huawei Zhang, and Caixia Gao. "CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture." Annual Review of Plant Biology 70, no. 1 (April 29, 2019): 667–97. http://dx.doi.org/10.1146/annurev-arplant-050718-100049.

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Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genome editing with crop breeding. We summarize the applications of genome editing for trait improvement, development of techniques for fine-tuning gene regulation, strategies for breeding virus resistance, and the use of high-throughput mutant libraries. We outline future perspectives for genome editing in plant synthetic biology and domestication, advances in delivery systems, editing specificity, homology-directed repair, and gene drives. Finally, we discuss the challenges and opportunities for precision plant breeding and its bright future in agriculture.
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Dissertations / Theses on the topic "Plant genome editing"

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Modrzejewski, Dominik [Verfasser]. "Evidence synthesis on the impact of genome editing on plant breeding / Dominik Modrzejewski." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1222738201/34.

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Gunadi, Andika. "Advancing CRISPR Applications Using Soybean [Glycine max (L.) Merr.] Promoters." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566169449003179.

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Tromp, Malou. "The environmental impact of introducing a potato protein for human consumption in Sweden." Thesis, Uppsala universitet, Institutionen för geovetenskaper, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-413595.

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In this study, a Consequential Life Cycle Assessment (CLCA) was conducted on the introduction of a potato protein for human consumption in Sweden. The assessed environmental impact cathegories in the CLCA were the categories global warming potential, eutrophication and land use. Potato protein is a side-stream that occurs during the production of potato starch and is currently used for animal feed (feed-grade). With the use of the new gene-editing technique CRISPR-Cas9, the stability of proteins in a starch potato can be improved to make the potato protein fit for human consumption (food-grade). The food-grade potato protein can be used as an ingredient in the food products: plant-based meat, quiche, sauces, wine and smoothies. When using the potato protein in one of these food products seven protein sources could potentially be substituted: soybean protein, yellow pea protein, beef protein, pork protein, chicken protein, egg protein and milk protein. The results of the CLCA show that when using the potato protein as an ingredient in a food product instead of other protein sources environmental impact can potentially be reduced. Most environmental impact can be reduced by substituting animal proteins by the potato protein. Therefore, from an environmental point of view, the most interesting food products to use the potato protein in as an ingredient are the food products where currently animal products are used in as the main source of protein.
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Zhang, Yingxiao. "Genetic Engineering of Rubber Producing Dandelions." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480626773100647.

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Leaser, Eileen Joanne. "TALENs: Site-Specific Genome Editing in Plants." Thesis, The University of Arizona, 2014. http://hdl.handle.net/10150/321773.

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Limones, Méndez Mariana Cecilia. "Développement d’outils moléculaires et cellulaires pour générer des variétés de Pomelo « Star Ruby » ne produisant pas de Furocoumarines." Thesis, Université de Lorraine, 2019. http://www.theses.fr/2019LORR0045.

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Les furocoumarines sont des composés phénoliques impliqués dans la défense contre les herbivores. Ces molécules sont majoritairement décrites dans quatre familles botaniques, notamment les Rutaceae, dont font partie les agrumes. Ces molécules sont phototoxiques ce qui peut poser des problèmes pour leur utilisation comme par exemple en cosmétique ou en phytothérapie. D’autre part, en cas d’ingestion par exemple via la consommation de jus de certains agrumes, elles ont responsables de l’inhibition d’enzymes de détoxication comme le CYP3A4 humain. Cela peut conduire à des surdosages médicamenteux connus sous le nom d’Effet Pomelo. Ce travail de thèse a consisté à réfléchir et à développer, des outils qui permettront de générer de manière ciblée des variétés de pomelo qui ne produisent plus de furocoumarines. Nous avons abordé l’ensemble des étapes essentielles pour la mise en place d’une stratégie global : i) des méthodes reproductibles ont été développées pour la production de protoplastes et de cultures cellulaires de pomelo Star Ruby ; ii) des conditions de transformation de protoplastes par électroporation ont également été mises au point ; iii) finalement, pour inhiber de manière spécifique la voie de biosynthèse des furocoumarines, nous avons choisi de mettre en œuvre une approche d’édition de génome en utilisant une méthodologie CRISPR/Cas9. La mise au point de la méthode a été réalisée avec un gène codant pour une umbelliferone 6-dimethylallyl transférase. Les résultats obtenus indiquent que la stratégie est envisageable. Pour renforcer la stratégie CRISPR/Cas9, nous avons mis en œuvre une démarche d’identification de gènes cibles additionnels. En utilisant une approche de data mining de bases de données génomiques et transcriptomiques nous avons identifié 18 séquences candidates, potentiellement impliquées dans la voie de biosynthèse des furocoumarines. L’expression hétérologue des protéines correspondantes et leur caractérisation fonctionnelle a permis de montrer que CYP706J12 est en mesure de métaboliser l’hérniarine, une coumarine. Ce résultat apporte des éléments pour émettre des hypothèses sur l’évolution convergente de la synthèse des coumarines et des furocoumarines chez les végétaux supérieurs
Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants
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Arndell, Taj. "Genome editing in wheat with CRISPR/Cas9." Thesis, 2019. http://hdl.handle.net/2440/120690.

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Genetically engineered crops have the potential to play a key role in achieving global food security and transitioning to a more sustainable agriculture. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for crop genome editing. CRISPR/Cas9 enables the targeted and precise modification of plant genomes via the creation and subsequent repair of site-specific DNA double-strand breaks (DSBs). The system consists of the Cas9 endonuclease in complex with a small guide RNA (gRNA) that is designed to target a specific site in the genome. Site-specific DSBs generated by Cas9 are repaired through non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is error-prone and often produces small insertions or deletions (indels) that result in gene knockout. Alternatively, if an exogenous DNA donor template is delivered to the cell, then precise modifications can be made via HDR. The CRISPR/Cas9 system has been successfully applied to many model and crop plants. However, it can be difficult to achieve highly efficient and specific editing in polyploid species. Therefore, the main aim of this PhD project was to develop tools and methods for optimising the CRISPR/Cas9 for efficient and specific genome editing in hexaploid bread wheat (Triticum aestivum). To test the efficacy of the CRISPR/Cas9 system for gene knockout, three gRNAs were designed to target Ms1, a male fertility gene that has been proposed for use in hybrid seed production. CRISPR/Cas9 vectors were delivered to immature embryos via Agrobacterium-mediated stable transformation, and the regenerated T0 lines were screened for targeted indels produced via NHEJ. Only one of the three gRNAs was efficacious. Five per cent (2/40) of T0 lines carrying the active gRNA were edited and male sterile, whereas all unedited lines were fully fertile. The recessive mutations were stably transmitted to the T1, T2 and T3 generations, as was the male sterile phonotype. Given the observed variability in the efficacy of different gRNAs targeting the same gene, and given that wheat transformation and tissue culture takes months and is laborious, a method was developed for the rapid assessment of gRNA activity and specificity. Seven gRNAs were designed to target EPSPS, a gene involved in aromatic amino acid biosynthesis. CRISPR/Cas9 vectors were then transiently transformed into wheat protoplasts. Three out of the seven gRNAs induced mutations at moderate to high frequencies. gRNA specificity was correlated with the number and distribution of mismatches in the ‘seed’ region of the gRNA. One of the gRNAs was selected as potentially suitable for the development of non-transgenic herbicide resistant wheat lines.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2019
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Modrzejewski, Dominik. "Evidence synthesis on the impact of genome editing on plant breeding." Thesis, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-1506-D.

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Bernabé, Orts Juan Miguel. "Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch." Doctoral thesis, 2019. http://hdl.handle.net/10251/133055.

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[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31.
[CAT] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31.
[EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch.
Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055
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Piatek, Agnieszka Anna. "Targeted Genome Regulation and Editing in Plants." Diss., 2016. http://hdl.handle.net/10754/606854.

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The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.
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Books on the topic "Plant genome editing"

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Qi, Yiping, ed. Plant Genome Editing with CRISPR Systems. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-8991-1.

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Dederer, Hans-Georg, and David Hamburger, eds. Regulation of Genome Editing in Plant Biotechnology. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-17119-3.

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Gupta, Om Prakash, and Suhas Gorakh Karkute. Genome Editing in Plants. Boca Raton: CRC Press, 2021. http://dx.doi.org/10.1201/9780367815370.

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Sivasankar, Shoba, Noel Ellis, Ljupcho Jankuloski, and Ivan Ingelbrecht, eds. Mutation breeding, genetic diversity and crop adaptation to climate change. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789249095.0000.

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Abstract This book presents reviews on the application of the technology for crop improvement towards food and nutrition security, and research status on mutation breeding and associated biotechnologies in both seed crops and vegetatively propagated crops. It also presents perspectives on the significance of next-generation sequencing and bioinformatics in determining the molecular variants underlying mutations and on emerging biotechnologies such as gene editing. Reviews and articles are organized into five sections in the publication: (1) Contribution of Crop Mutant Varieties to Food Security; (2) Mutation Breeding in Crop Improvement and Climate-Change Adaptation; (3) Mutation Induction Techniques for Enhanced Genetic Variation; (4) Mutation Breeding in Vegetatively Propagated and Ornamental Crops; and (5) Induced Genetic Variation for Crop Improvement in the Genomic Era. The contents of this volume present excellent reference material for researchers, students and policy makers involved in the application of induced genetic variation in plants for the maintenance of biodiversity and the acceleration of crop adaptation to climate change to feed a growing global population in the coming years and decades.
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Sprink, Thorben, Ralf Alexander Wilhelm, Armin Spök, Jürgen Robienski, Stephan Schleissing, and Joachim Hermann Schiemann, eds. Plant Genome Editing – Policies and Governance. Frontiers Media SA, 2020. http://dx.doi.org/10.3389/978-2-88963-670-9.

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Dürnberger, Christian, Sebastian Pfeilmeier, and Stephan Schleissing, eds. Genome Editing in Agriculture. Nomos Verlagsgesellschaft mbH & Co. KG, 2019. http://dx.doi.org/10.5771/9783845296432.

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New plant breeding techniques such as CRISPR/Cas have the potential to improve sustainability in agriculture. Genome editing techniques can increase yields while reducing the use of pesticides. Researchers around the world are working on improving the nutritional value of plants. However, whether the new technologies will be used in Europe is uncertain at present. Should genome editing be regulated like the ‘old’ genetic engineering techniques used on plants? What might a responsible interpretation of the precautionary principle look like? The political discussion on the evaluation of new plant breeding technologies is in full swing. The contributions in this anthology present the legal, social and ethical aspects of the topic that were discussed at a summer school of the Institute of Technology-Theology-Natural Sciences (TTN) at Ludwig Maximilian University in Munich. With contributions from Stephan Schleissing; Sebastian Pfeilmeier; Christian Dürnberger; Jarst van Belle; Jan Schaart; Robert van Loo; Katharina Unkel; Thorben Sprink; Aurélie Jouanin; Marinus J.M. Smulders; Hans-Georg Dederer; Brigitte Voigt; Felix Beck; João Otávio Benevides Demasi; Bartosz Bartkowski; Chad M. Baum; Alexander Bogner; Helge Torgersen; Sebastian Schubert; Anne Friederike Hoffmann; Ksenia Gerasimova; Karolina Rucinska
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Crop Biotechnology: Genetic Modification and Genome Editing. World Scientific Publishing Co Pte Ltd, 2018.

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Qi, Yiping. Plant Genome Editing with CRISPR Systems: Methods and Protocols. Springer New York, 2019.

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Deka, Pradip Chandra. Molecular Plant Breeding and Genome Editing Tools for Crop Improvement. IGI Global, 2020.

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Deka, Pradip Chandra. Molecular Plant Breeding and Genome Editing Tools for Crop Improvement. IGI Global, 2020.

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Book chapters on the topic "Plant genome editing"

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Wada, Naoki, Yuriko Osakabe, and Keishi Osakabe. "Plant Genome Editing." In Plant Omics, 205–16. GB: CABI, 2022. http://dx.doi.org/10.1079/9781789247534.0015.

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Eriksson, Dennis, and Leire Escajedo San-Epifanio. "Plant Genome Editing Governance." In Encyclopedia of Food and Agricultural Ethics, 1–5. Dordrecht: Springer Netherlands, 2018. http://dx.doi.org/10.1007/978-94-007-6167-4_637-1.

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Eriksson, Dennis, and Leire Escajedo San-Epifanio. "Plant Genome Editing Governance." In Encyclopedia of Food and Agricultural Ethics, 1980–85. Dordrecht: Springer Netherlands, 2019. http://dx.doi.org/10.1007/978-94-024-1179-9_637.

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Kumar, Gaurav, Bhupendra Singh Panwar, and Nabaneeta Basak. "Genome Editing in Plant." In Genome Editing in Plants, 203–15. Boca Raton: CRC Press, 2021. http://dx.doi.org/10.1201/9780367815370-14.

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Nishitani, Chikako, Keishi Osakabe, and Yuriko Osakabe. "Genome Editing in Apple." In Compendium of Plant Genomes, 213–25. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-74682-7_10.

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Butler, Nathaniel M., Jiming Jiang, and Robert M. Stupar. "Crop Improvement Using Genome Editing." In Plant Breeding Reviews, 55–101. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2018. http://dx.doi.org/10.1002/9781119414735.ch2.

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Hundleby, Penny, and Wendy Harwood. "Regulatory Constraints and Differences of Genome-Edited Crops Around the Globe." In Genome Editing, 319–41. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-08072-2_17.

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AbstractPlant breeding for centuries has relied on the availability of genetic variation to introduce new desirable traits into crops. Biotechnology has already accelerated the ability to induce and utilize new genetic variation, through approaches such as mutation breeding and using technologies such as marker assisted breeding to rapidly identify the required variation. These technologies fall within the definition of “conventional and traditional” breeding and are lightly regulated. However, plant breeders are facing an urgent need for access to wider genetic variation to meet the needs of today’s farmers and consumers worldwide. New breeding technologies (NBTs), such as genome editing, are speeding up the breeding process and providing plant breeders with access to a far greater range of genetic variation. Coupled with a rapidly accelerating genomics era, genome editing is moving plant breeding into an exciting era of intelligent and precision-based plant breeding. The speed at which these new technologies are emerging has challenged the regulatory climate. Some countries consider genome edited crops to require the same regulatory oversight as genetically modified organisms (GMOs), while others have chosen to regulate with the same safety evaluations currently associated with bringing conventionally bred crops to market. Harmonization of the regulatory climate is urgently needed if there is to be equal access to this technology and to support international trade of these crops. The current chapter provides a global overview of the current regulatory status of genome-edited crops.
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Mehta, D. R., and A. K. Nandha. "Genome Editing in Plant Breeding." In Advanced Molecular Plant Breeding, 605–44. Toronto ; New Jersey : Apple Academic Press, 2018.: Apple Academic Press, 2018. http://dx.doi.org/10.1201/b22473-18.

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Qin, Ruiying, and Pengcheng Wei. "Plant Precise Genome Editing by Prime Editing." In Genome Editing Technologies for Crop Improvement, 177–83. Singapore: Springer Nature Singapore, 2022. http://dx.doi.org/10.1007/978-981-19-0600-8_9.

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Witzany, Günther. "Plant Communication." In Biocommunication and Natural Genome Editing, 27–51. Dordrecht: Springer Netherlands, 2009. http://dx.doi.org/10.1007/978-90-481-3319-2_2.

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Conference papers on the topic "Plant genome editing"

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Rastogi, Khushboo. "Rice Biofortification through CRISPR/Cas9-Multiplex Genome Editing." In ASPB PLANT BIOLOGY 2020. USA: ASPB, 2020. http://dx.doi.org/10.46678/pb.20.1383191.

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"Genome editing in wheat: exploration of new challenges for crop improvement." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-096.

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"CRISPR/Cas9 – mediated genome editing of bread wheat to modulate heading time." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-135.

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"Improvement of sorghum seed storage protein digestibility using RNA-interference and genome editing." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-048.

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Kershanskaya, O. I., G. S. Mukiyanova, D. S. Nelidova, G. L. Esenbaeva, S. N. Nelidov, K. R. Uteulin, and J. Stephens. "CRISPR/Cas9 editing the genome of crops in the development of biology and agriculture." In IX Congress of society physiologists of plants of Russia "Plant physiology is the basis for creating plants of the future". Kazan University Press, 2019. http://dx.doi.org/10.26907/978-5-00130-204-9-2019-207.

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"Comparative assessment of sugar accumulation in commercial potato cultivars (Solanum tuberosum L.) for genome editing." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-175.

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Stacey, Minviluz. "Utility of CRISPR/Cas in accelerating gene discovery in soybean." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/rzne1660.

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The use of CRISPR/Cas9 has been successfully applied in various plant species to induce targeted genome editing, including soybean. Soybean is recalcitrant to transformation and thus, plants with stable CRISPR gene edits are costly and take a long time to produce. Moreover, soybean is allotetraploid and editing paralogous genes are often necessary to obtain observable phenotype(s). For each gene target, we designed two gRNAs to increase editing efficiency and allow rapid genotyping by PCR. We also tested the CRISPR reagents in transient hairy root transformation to determine if the Cas9 and gRNAs would perform properly in transgenic soybean plants. Our results showed that we can indeed obtain highly efficient, cost-effective CRISPR/Cas editing in soybean to generate novel genotypes for gene discovery and downstream field propagation and breeding efforts. Examples of CRISPR-edited genes and their associated seed traits will be discussed.
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"Redesign of starch biosynthetic pathway in rice by CRISPR/Cas9-mediated genome editing toward human diets." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-074.

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"Applications of the CRISPR/Cas9 genome editing system for modification of starch content in wheat and triticale." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-197.

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"New breakthrough CRISPR/Cas9 biotechnology of genome editing is a powerful tool for improvement of agricultural crops." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-080.

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Reports on the topic "Plant genome editing"

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Wilson, Thomas E., Avraham A. Levy, and Tzvi Tzfira. Controlling Early Stages of DNA Repair for Gene-targeting Enhancement in Plants. United States Department of Agriculture, March 2012. http://dx.doi.org/10.32747/2012.7697124.bard.

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Gene targeting (GT) is a much needed technology as a tool for plant research and for the precise engineering of crop species. Recent advances in this field have shown that the presence of a DNA double-strand break (DSB) in a genomic locus is critical for the integration of an exogenous DNA molecule introduced into this locus. This integration can occur via either non-homologous end joining (NHEJ) into the break or homologous recombination (HR) between the broken genomic DNA and the introduced vector. A bottleneck for DNA integration via HR is the machinery responsible for homology search and strand invasion. Important proteins in this pathway are Rad51, Rad52 and Rad54. We proposed to combine our respective expertise: on the US side, in the design of zincfinger nucleases (ZFNs) for the induction of DNA DSBs at any desired genomic locus and in the integration of DNA molecules via NHEJ; and on the Israeli side in the HR events, downstream of the DSB, that lead to homology search and strand invasion. We sought to test three major pathways of targeted DNA integration: (i) integration by NHEJ into DSBs induced at desired sites by specially designed ZFNs; (ii) integration into DSBs induced at desired sites combined with the use of Rad51, Rad52 and Rad54 proteins to maximize the chances for efficient and precise HR-mediated vector insertion; (iii) stimulation of HR by Rad51, Rad52 and Rad54 in the absence of DSB induction. We also proposed to study the formation of dsT-DNA molecules during the transformation of plant cells. dsT-DNA molecules are an important substrate for HR and NHEJ-mediatedGT, yet the mode of their formation from single stranded T-DNA molecules is still obscure. In addition we sought to develop a system for assembly of multi-transgene binary vectors by using ZFNs. The latter may facilitate the production of binary vectors that may be ready for genome editing in transgenic plants. ZFNs were proposed for the induction of DSBs in genomic targets, namely, the FtsH2 gene whose loss of function can easily be identified in somatic tissues as white sectors, and the Cruciferin locus whose targeting by a GFP or RFP reporter vectors can give rise to fluorescent seeds. ZFNs were also proposed for the induction of DSBs in artificial targets and for assembly of multi-gene vectors. We finally sought to address two important cell types in terms of relevance to plant transformation, namely GT of germinal (egg) cells by floral dipping, and GT in somatic cells by root and leave transformation. To be successful, we made use of novel optimized expression cassettes that enable coexpression of all of the genes of interest (ZFNs and Rad genes) in the right tissues (egg or root cells) at the right time, namely when the GT vector is delivered into the cells. Methods were proposed for investigating the complementation of T-strands to dsDNA molecules in living plant cells. During the course of this research, we (i) designed, assembled and tested, in vitro, a pair of new ZFNs capable of targeting the Cruciferin gene, (ii) produced transgenic plants which expresses for ZFN monomers for targeting of the FtsH2 gene. Expression of these enzymes is controlled by constitutive or heat shock induced promoters, (iii) produced a large population of transgenic Arabidopsis lines in which mutated mGUS gene was incorporated into different genomic locations, (iv) designed a system for egg-cell-specific expression of ZFNs and RAD genes and initiate GT experiments, (v) demonstrated that we can achieve NHEJ-mediated gene replacement in plant cells (vi) developed a system for ZFN and homing endonuclease-mediated assembly of multigene plant transformation vectors and (vii) explored the mechanism of dsTDNA formation in plant cells. This work has substantially advanced our understanding of the mechanisms of DNA integration into plants and furthered the development of important new tools for GT in plants.
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Wentworth, Jonathan, and David Rapley. Genome edited animals. Parliamentary Office of Science and Technology, November 2022. http://dx.doi.org/10.58248/pb50.

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Genome editing, also known as gene editing, encompasses a broad range of techniques that allows targeted changes in the DNA of animals (and plants). The Genetic Technology (Precision Breeding) Bill 2022 -2023, due for Second Reading in the House of Lords on 21 November 2022, intends to change the regulatory definition of certain genome-edited animals.
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Brown Horowitz, Sigal, Eric L. Davis, and Axel Elling. Dissecting interactions between root-knot nematode effectors and lipid signaling involved in plant defense. United States Department of Agriculture, January 2014. http://dx.doi.org/10.32747/2014.7598167.bard.

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Root-knot nematodes, Meloidogynespp., are extremely destructive pathogens with a cosmopolitan distribution and a host range that affects most crops. Safety and environmental concerns related to the toxicity of nematicides along with a lack of natural resistance sources threaten most crops in Israel and the U.S. This emphasizes the need to identify genes and signal mechanisms that could provide novel nematode control tactics and resistance breeding targets. The sedentary root-knot nematode (RKN) Meloidogynespp. secrete effectors in a spatial and temporal manner to interfere with and mimic multiple physiological and morphological mechanisms, leading to modifications and reprogramming of the host cells' functions, resulted in construction and maintenance of nematodes' feeding sites. For successful parasitism, many effectors act as immunomodulators, aimed to manipulate and suppress immune defense signaling triggered upon nematode invasion. Plant development and defense rely mainly on hormone regulation. Herein, a metabolomic profiling of oxylipins and hormones composition of tomato roots were performed using LC-MS/MS, indicating a fluctuation in oxylipins profile in a compatible interaction. Moreover, further attention was given to uncover the implication of WRKYs transcription factors in regulating nematode development. In addition, in order to identify genes that might interact with the lipidomic defense pathway induced by oxylipins, a RNAseq was performed by exposing M. javanicasecond-stage juveniles to tomato protoplast, 9-HOT and 13-KOD oxylipins. This transcriptome generated a total of 4682 differentially expressed genes (DEGs). Being interested in effectors, we seek for DEGs carrying a predicted secretion signal peptide. Among the DEGs including signal peptide, several had homology with known effectors in other nematode species, other unknown potentially secreted proteins may have a role as root-knot nematodes' effectors which might interact with lipid signaling. The molecular interaction of LOX proteins with the Cyst nematode effectors illustrate the nematode strategy in manipulating plant lipid signals. The function of several other effectors in manipulating plant defense signals, as well as lipids signals, weakening cell walls, attenuating feeding site function and development are still being studied in depth for several novel effectors. As direct outcome of this project, the accumulating findings will be utilized to improve our understanding of the mechanisms governing critical life-cycle phases of the parasitic M. incognita RKN, thereby facilitating design of effective controls based on perturbation of nematode behavior—without producing harmful side effects. The knowledge from this study will promote genome editing strategies aimed at developing nematode resistance in tomato and other nematode-susceptible crop species in Israel and the United States.
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Snell, Kristi. Production of high oil, transgene free Camelina sativa plants through genome editing. Office of Scientific and Technical Information (OSTI), December 2018. http://dx.doi.org/10.2172/1489126.

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Li, Li, Joseph Burger, Nurit Katzir, Yaakov Tadmor, Ari Schaffer, and Zhangjun Fei. Characterization of the Or regulatory network in melon for carotenoid biofortification in food crops. United States Department of Agriculture, April 2015. http://dx.doi.org/10.32747/2015.7594408.bard.

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The general goals of the BARD research grant US-4423-11 are to understand how Or regulates carotenoid accumulation and to reveal novel strategies for breeding agricultural crops with enhanced β-carotene level. The original objectives are: 1) to identify the genes and proteins in the Or regulatory network in melon; 2) to genetically and molecularly characterize the candidate genes; and 3) to define genetic and functional allelic variation of these genes in a representative germplasm collection of the C. melo species. Or was found by the US group to causes provitamin A accumulation in chromoplasts in cauliflower. Preliminary genetic study from the Israeli group revealed that the melon Or gene (CmOr) completely co-segregated with fruit flesh color in a segregating mapping population and in a wide melon germplasm collection, which set the stage for the funded research. Major conclusions and achievements include: 1). CmOris proved to be the gene that controls melon fruit flesh color and represents the previously described gflocus in melon. 2). Genetic and molecular analyses of CmOridentify and confirm a single SNP that is responsible for the orange and non-orange phenotypes in melon fruit. 3). Alteration of the evolutionarily conserved arginine in an OR protein to both histidine or alanine greatly enhances its ability to promote carotenoid accumulation. 4). OR promotes massive carotenoid accumulation due to its dual functions in regulating both chromoplast biogenesis and carotenoid biosynthesis. 5). A bulk segregant transcriptome (BSRseq) analysis identifies a list of genes associated with the CmOrregulatory network. 6). BSRseq is proved to be an effective approach for gene discovery. 7). Screening of an EMS mutation library identifies a low β mutant, which contains low level of carotenoids due to a mutation in CmOrto produce a truncated form of OR protein. 8). low β exhibits lower germination rate and slow growth under salt stress condition. 9). Postharvest storage of fruit enhances carotenoid accumulation, which is associated with chromoplast development. Our research uncovers the molecular mechanisms underlying the Or-regulated high level of carotenoid accumulation via regulating carotenoidbiosynthetic capacity and storage sink strength. The findings provide mechanistic insights into how carotenoid accumulation is controlled in plants. Our research also provides general and reliable molecular markers for melon-breeding programs to select orange varieties, and offers effective genetic tools for pro-vitamin A enrichment in other important crops via the rapidly developed genome editing technology. The newly discovered low β mutant could lead to a better understanding of the Or gene function and its association with stress response, which may explain the high conservation of the Or gene among various plant species.
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Schuster, Gadi, and David Stern. Integrated Studies of Chloroplast Ribonucleases. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7697125.bard.

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Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.
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