The Experts below are selected from a list of 115962 Experts worldwide ranked by ideXlab platform
Daniel F. Voytas - One of the best experts on this subject based on the ideXlab platform.
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targeted mutagenesis in Plant cells through transformation of sequence specific nuclease mrna
PLOS ONE, 2016Co-Authors: Thomas Stoddard, Daniel F. Voytas, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Feng Zhang, Song LuoAbstract:Plant Genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied Plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the Plant Genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to Plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of Genome editing.
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a crispr cas9 toolbox for multiplexed Plant Genome editing and transcriptional regulation
Plant Physiology, 2015Co-Authors: Levi G Lowder, Daniel F. Voytas, Nicholas J Baltes, Dengwei Zhang, Joseph W Paul, Xu Tang, Xuelian Zheng, Tzungfu Hsieh, Yong ZhangAbstract:The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In Plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant Plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other Genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for Plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in Plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of Plant endogenous genes. We report the functionality and effectiveness of this toolbox in model Plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied Plant research.
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non transgenic Plant Genome editing using purified sequence specific nucleases
Molecular Plant, 2015Co-Authors: Song Luo, Daniel F. Voytas, Thomas Stoddard, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Andrew Coffman, Adam Retterath, Luc Mathis, Feng ZhangAbstract:Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs), and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of Plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host Genome.
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dna replicons for Plant Genome engineering
The Plant Cell, 2014Co-Authors: Nicholas J Baltes, Tomas Cermak, Javier Gilhumanes, Paul Atkins, Daniel F. VoytasAbstract:Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of Plant Genomes remains challenging due to ineffective methods for delivering reagents for Genome engineering to Plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate Plants with a desired DNA sequence modification. By adopting a general Plant transformation method, Plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for Plant Genome engineering.
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Plant Genome engineering with sequence specific nucleases
Annual Review of Plant Biology, 2013Co-Authors: Daniel F. VoytasAbstract:Recent advances in Genome engineering provide newfound control over a Plant's genetic material. It is now possible for most bench scientists to alter DNA in living Plant cells in a variety of ways, including introducing specific nucleotide substitutions in a gene that change a protein's amino acid sequence, deleting genes or chromosomal segments, and inserting foreign DNA at precise genomic locations. Such targeted DNA sequence modifications are enabled by sequence-specific nucleases that create double-strand breaks in the genomic loci to be altered. The repair of the breaks, through either homologous recombination or nonhomologous end joining, can be controlled to achieve the desired sequence modification. Genome engineering promises to advance basic Plant research by linking DNA sequences to biological function. Further, Genome engineering will enable Plants' biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population.
Klaus F X Mayer - One of the best experts on this subject based on the ideXlab platform.
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pgsb Plantsdb updates to the database framework for comparative Plant Genome research
Nucleic Acids Research, 2016Co-Authors: Manuel Spannagl, Heidrun Gundlach, Thomas Nussbaumer, Kai Christian Bader, Mihaela Martis, Michael Seidel, Karl G Kugler, Klaus F X MayerAbstract:PGSB (Plant Genome and Systems Biology: formerly MIPS) PlantsDB (http://pgsb.helmholtz-muenchen.de/Plant/index.jsp) is a database framework for the comparative analysis and visualization of Plant Genome data. The resource has been updated with new data sets and types as well as specialized tools and interfaces to address user demands for intuitive access to complex Plant Genome data. In its latest incarnation, we have re-worked both the layout and navigation structure and implemented new keyword search options and a new BLAST sequence search functionality. Actively involved in corresponding sequencing consortia, PlantsDB has dedicated special efforts to the integration and visualization of complex triticeae Genome data, especially for barley, wheat and rye. We enhanced CrowsNest, a tool to visualize syntenic relationships between Genomes, with data from the wheat sub-Genome progenitor Aegilops tauschii and added functionality to the PGSB RNASeqExpressionBrowser. GenomeZipper results were integrated for the Genomes of barley, rye, wheat and perennial ryegrass and interactive access is granted through PlantsDB interfaces. Data exchange and cross-linking between PlantsDB and other Plant Genome databases is stimulated by the transPlant project (http://transPlantdb.eu/).
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Plant Genome sequencing - applications for crop improvement
Current Opinion in Biotechnology, 2014Co-Authors: Marie E. Bolger, Uwe Scholz, Bernd Weisshaar, Bjorn Usadel, Klaus F X MayerAbstract:It is over 10 years since the Genome sequence of the first crop was published. Since then, the number of crop Genomes sequenced each year has increased steadily. The amazing pace at which Genome sequences are becoming available is largely due to the improvement in sequencing technologies both in terms of cost and speed. Modern sequencing technologies allow the sequencing of multiple cultivars of smaller crop Genomes at a reasonable cost. Though many of the published Genomes are considered incomplete, they nevertheless have proved a valuable tool to understand important crop traits such as fruit ripening, grain traits and flowering time adaptation. © 2013.
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MIPSPlantsDB--Plant database resource for integrative and comparative Plant Genome research.
Nucleic acids research, 2007Co-Authors: Manuel Spannagl, Heidrun Gundlach, Octave Noubibou, Dirk Haase, Tobias Hindemitt, Kathrin Klee, Georg Haberer, Heiko Schoof, Li Yang, Klaus F X MayerAbstract:Genome-oriented Plant research delivers rapidly increasing amount of Plant Genome data. Comprehensive and structured information resources are required to structure and communicate Genome and associated analytical data for model organisms as well as for crops. The increase in available Plant genomic data enables powerful comparative analysis and integrative approaches. PlantsDB aims to provide data and information resources for individual Plant species and in addition to build a platform for integrative and comparative Plant Genome research. PlantsDB is constituted from Genome databases for Arabidopsis, Medicago, Lotus, rice, maize and tomato. Complementary data resources for cis elements, repetive elements and extensive cross-species comparisons are implemented. The PlantsDB portal can be reached at http://mips.gsf.de/projects/Plants.
John B Ohlrogge - One of the best experts on this subject based on the ideXlab platform.
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toward a functional catalog of the Plant Genome a survey of genes for lipid biosynthesis
Plant Physiology, 2000Co-Authors: Sergei Mekhedov, Oskar Martinez De Ilarduya, John B OhlroggeAbstract:Public databases now include vast amounts of recently acquired DNA sequences that are only partially annotated and, furthermore, are often annotated by automated methods that are subject to errors. Maximum information value of these databases can be derived only by further detailed analyses that frequently require careful examination of records in the context of biological functions. In this study we present an example of such an analysis focused on Plant glycerolipid synthesis. Public databases were searched for sequences corresponding to 65 Plant polypeptides involved in lipid metabolism. Comprehensive search results and analysis of genes, cDNAs and expressed sequence tags (ESTs) are available online (http://www.canr.msu.edu/lgc). Multiple alignments provided a method to estimate the number of genes in gene families. Further analysis of sequences allowed us to tentatively identify several previously undescribed genes in Arabidopsis. For example, two genomic sequences were identified as candidates for the palmitate-specific monogalactosyldiacylglycerol desaturase (FAD5). A candidate genomic sequence for 3-ketoacyl-acyl-carrier protein (ACP) synthase involved in mitochondrial fatty acid biosynthesis was also identified. Biotin carboxyl carrier protein (BCCP) in Arabidopsis is encoded by at least two genes, but the most abundant BCCP transcript so far has not been characterized. The large number (>165,000) of Plant ESTs also provides an opportunity to perform “digital northern” comparisons of gene expression levels across many genes. EST abundance in general correlated with biochemical and flux characteristics of the enzymes in Arabidopsis leaf tissue. In a few cases, statistically significant differences in EST abundance levels were observed for enzymes that catalyze similar reactions in fatty acid metabolism. For example, ESTs for the FatB acyl-ACP thioesterase occur 21 times compared with 7 times for FatA acyl-ACP thioesterase, although flux through the FatA reaction is several times higher than through FatB. Such comparisons may provide initial clues toward previously undescribed regulatory phenomena. The abundance of ESTs for ACP compared with that of stearoyl-ACP desaturase and FatB acyl-ACP thioesterase suggests that concentrations of some enzymes of fatty acid synthesis may be higher than their acyl-ACP substrates.
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toward a functional catalog of the Plant Genome a survey of genes for lipid biosynthesis
Plant Physiology, 2000Co-Authors: Sergei Mekhedov, Oskar Martinez De Ilarduya, John B OhlroggeAbstract:Public databases now include vast amounts of recently acquired DNA sequences that are only partially annotated and, furthermore, are often annotated by automated methods that are subject to errors. Maximum information value of these databases can be derived only by further detailed analyses that frequently require careful examination of records in the context of biological functions. In this study we present an example of such an analysis focused on Plant glycerolipid synthesis. Public databases were searched for sequences corresponding to 65 Plant polypeptides involved in lipid metabolism. Comprehensive search results and analysis of genes, cDNAs and expressed sequence tags (ESTs) are available online (http://www.canr.msu.edu/lgc). Multiple alignments provided a method to estimate the number of genes in gene families. Further analysis of sequences allowed us to tentatively identify several previously undescribed genes in Arabidopsis. For example, two genomic sequences were identified as candidates for the palmitate-specific monogalactosyldiacylglycerol desaturase (FAD5). A candidate genomic sequence for 3-ketoacyl-acyl-carrier protein (ACP) synthase involved in mitochondrial fatty acid biosynthesis was also identified. Biotin carboxyl carrier protein (BCCP) in Arabidopsis is encoded by at least two genes, but the most abundant BCCP transcript so far has not been characterized. The large number (>165,000) of Plant ESTs also provides an opportunity to perform “digital northern” comparisons of gene expression levels across many genes. EST abundance in general correlated with biochemical and flux characteristics of the enzymes in Arabidopsis leaf tissue. In a few cases, statistically significant differences in EST abundance levels were observed for enzymes that catalyze similar reactions in fatty acid metabolism. For example, ESTs for the FatB acyl-ACP thioesterase occur 21 times compared with 7 times for FatA acyl-ACP thioesterase, although flux through the FatA reaction is several times higher than through FatB. Such comparisons may provide initial clues toward previously undescribed regulatory phenomena. The abundance of ESTs for ACP compared with that of stearoyl-ACP desaturase and FatB acyl-ACP thioesterase suggests that concentrations of some enzymes of fatty acid synthesis may be higher than their acyl-ACP substrates.
Yong Zhang - One of the best experts on this subject based on the ideXlab platform.
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the improvement of crispr cas9 system with ubiquitin associated domain fusion for efficient Plant Genome editing
Frontiers in Plant Science, 2020Co-Authors: Xuelian Zheng, Zhaohui Zhong, Lijia Yang, Xu Tang, Quan Quan, Binglin Liu, Tingting Fan, Jianping Zhou, Yong ZhangAbstract:Genome editing technology represented by CRISPR-Cas9 had been widely used in many biological fields such as gene function analysis, gene therapy, and crop improvement. However, in the face of the complexity of the eukaryotic Genome, the CRISPR-Cas9 Genome editing tools have shown an unstable editing efficiency with large variability at different target sites. It was important to further improve the editing efficiency of the CRISPR-Cas9 system among the whole Genome. In this study, based on the previous single transcription unit Genome editing system (STU-SpCas9), using the ubiquitin-associated domain (UBA) to enhance the stability of Cas9 protein, we constructed three Cas9-UBA fusion systems (SpCas9-SD01, SpCas9-SD02, and SpCas9-SD03). Four different target sites of rice OsPDS, OsDEP1 and OsROC5 genes were chosen to evaluate the Genome editing efficiency in rice protoplasts and stable transformed rice Plants. The results showed that the fusion of UBA domains did not affect the cleavage mode of Cas9 protein, and effectively increase the editing efficiency of STU-SpCas9 at the target sites. This new CRISPR-Cas9-UBA system provided a new strategy and tool for improving the Genome editing efficiency of CRISPR-Cas9 in Plants.
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a crispr cas9 toolbox for multiplexed Plant Genome editing and transcriptional regulation
Plant Physiology, 2015Co-Authors: Levi G Lowder, Daniel F. Voytas, Nicholas J Baltes, Dengwei Zhang, Joseph W Paul, Xu Tang, Xuelian Zheng, Tzungfu Hsieh, Yong ZhangAbstract:The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In Plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant Plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other Genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for Plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in Plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of Plant endogenous genes. We report the functionality and effectiveness of this toolbox in model Plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied Plant research.
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transcription activator like effector nucleases enable efficient Plant Genome engineering
Plant Physiology, 2013Co-Authors: Yong Zhang, Feng Zhang, Joshua A Baller, Colby G Starker, Adam J Bogdanove, Daniel F. VoytasAbstract:The ability to precisely engineer Plant Genomes offers much potential for advancing basic and applied Plant biology. Here, we describe methods for the targeted modification of Plant Genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover Genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in Plant protoplasts should be useful for targeted modification of cells from diverse Plant species and using a variety of means for reagent delivery.
Nicholas J Baltes - One of the best experts on this subject based on the ideXlab platform.
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targeted mutagenesis in Plant cells through transformation of sequence specific nuclease mrna
PLOS ONE, 2016Co-Authors: Thomas Stoddard, Daniel F. Voytas, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Feng Zhang, Song LuoAbstract:Plant Genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied Plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the Plant Genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to Plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of Genome editing.
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a crispr cas9 toolbox for multiplexed Plant Genome editing and transcriptional regulation
Plant Physiology, 2015Co-Authors: Levi G Lowder, Daniel F. Voytas, Nicholas J Baltes, Dengwei Zhang, Joseph W Paul, Xu Tang, Xuelian Zheng, Tzungfu Hsieh, Yong ZhangAbstract:The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In Plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant Plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other Genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for Plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in Plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of Plant endogenous genes. We report the functionality and effectiveness of this toolbox in model Plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied Plant research.
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non transgenic Plant Genome editing using purified sequence specific nucleases
Molecular Plant, 2015Co-Authors: Song Luo, Daniel F. Voytas, Thomas Stoddard, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Andrew Coffman, Adam Retterath, Luc Mathis, Feng ZhangAbstract:Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs), and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of Plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host Genome.
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dna replicons for Plant Genome engineering
The Plant Cell, 2014Co-Authors: Nicholas J Baltes, Tomas Cermak, Javier Gilhumanes, Paul Atkins, Daniel F. VoytasAbstract:Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of Plant Genomes remains challenging due to ineffective methods for delivering reagents for Genome engineering to Plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate Plants with a desired DNA sequence modification. By adopting a general Plant transformation method, Plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for Plant Genome engineering.
