62 results on '"Colby G Starker"'
Search Results
2. Building customizable auto-luminescent luciferase-based reporters in plants
- Author
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Arjun Khakhar, Colby G Starker, James C Chamness, Nayoung Lee, Sydney Stokke, Cecily Wang, Ryan Swanson, Furva Rizvi, Takato Imaizumi, and Daniel F Voytas
- Subjects
bioluminescence ,luciferase ,Nicotiana benthamiana ,hormone reporters ,expression reporter ,plant synthetic biology ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.
- Published
- 2020
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3. Correction: Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.
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František Liška, Renata Peterková, Miroslav Peterka, Vladimír Landa, Václav Zídek, Petr Mlejnek, Jan Šilhavý, Miroslava Šimáková, Vladimír Křen, Colby G Starker, Daniel F Voytas, Zsuzsanna Izsvák, and Michal Pravenec
- Subjects
Medicine ,Science - Abstract
[This corrects the article DOI: 10.1371/journal.pone.0164206.].
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- 2020
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- View/download PDF
4. Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana.
- Author
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Jia He, Mingli Xu, Matthew R Willmann, Kevin McCormick, Tieqiang Hu, Li Yang, Colby G Starker, Daniel F Voytas, Blake C Meyers, and R Scott Poethig
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Genetics ,QH426-470 - Abstract
Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.
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- 2018
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5. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.
- Author
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František Liška, Renata Peterková, Miroslav Peterka, Vladimír Landa, Václav Zídek, Petr Mlejnek, Jan Šilhavý, Miroslava Šimáková, Vladimír Křen, Colby G Starker, Daniel F Voytas, Zsuzsanna Izsvák, and Michal Pravenec
- Subjects
Medicine ,Science - Abstract
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.
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- 2016
- Full Text
- View/download PDF
6. TAL effector specificity for base 0 of the DNA target is altered in a complex, effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat -1.
- Author
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Erin L Doyle, Aaron W Hummel, Zachary L Demorest, Colby G Starker, Daniel F Voytas, Philip Bradley, and Adam J Bogdanove
- Subjects
Medicine ,Science - Abstract
TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5' base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.
- Published
- 2013
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7. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.
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Timothy J Dahlem, Kazuyuki Hoshijima, Michael J Jurynec, Derrick Gunther, Colby G Starker, Alexandra S Locke, Allison M Weis, Daniel F Voytas, and David Jonah Grunwald
- Subjects
Genetics ,QH426-470 - Abstract
The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.
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- 2012
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8. Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues.
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Michelle L Christian, Zachary L Demorest, Colby G Starker, Mark J Osborn, Michael D Nyquist, Yong Zhang, Daniel F Carlson, Philip Bradley, Adam J Bogdanove, and Daniel F Voytas
- Subjects
Medicine ,Science - Abstract
The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths - from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
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- 2012
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9. An extensible vector toolkit and parts library for advanced engineering of plant genomes
- Author
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James C. Chamness, Jitesh Kumar, Anna J. Cruz, Elissa Rhuby, Mason J. Holum, Jon P. Cody, Redeat Tibebu, Maria Elena Gamo, Colby G. Starker, Feng Zhang, and Daniel F. Voytas
- Subjects
Plant culture ,SB1-1110 ,Genetics ,QH426-470 - Abstract
Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.
- Published
- 2023
- Full Text
- View/download PDF
10. Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana
- Author
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Jon P. Cody, Michael F. Maher, Ryan A. Nasti, Colby G. Starker, James C. Chamness, and Daniel F. Voytas
- Subjects
Article ,General Biochemistry, Genetics and Molecular Biology - Abstract
There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.
- Published
- 2022
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11. Heritable base-editing in Arabidopsis using RNA viral vectors
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Degao Liu, Shuya Xuan, Lynn E Prichard, Lilee I Donahue, Changtian Pan, Ugrappa Nagalakshmi, Evan E Ellison, Colby G Starker, Savithramma P Dinesh-Kumar, Yiping Qi, and Daniel F Voytas
- Subjects
Arabidopsis Proteins ,RNA, Plant ,Physiology ,Mutation ,Arabidopsis ,Genetics ,RNA ,RNA Editing ,Plant Science - Abstract
Heritable base-editing using a viral delivery system enables high-throughput functional analysis of genes in Arabidopsis.
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- 2022
- Full Text
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12. An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes
- Author
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James C. Chamness, Jitesh Kumar, Anna J. Cruz, Elissa Rhuby, Mason J. Holum, Jon P. Cody, Redeat Tibebu, Maria Elena Gamo, Colby G. Starker, Feng Zhang, and Daniel F. Voytas
- Subjects
Genetics ,Plant Science ,Agronomy and Crop Science - Abstract
Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.
- Published
- 2022
- Full Text
- View/download PDF
13. Plant gene editing through de novo induction of meristems
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Macy Vollbrecht, Michael F. Maher, Matthew D. Clark, Colby G. Starker, Daniel F. Voytas, and Ryan A. Nasti
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0303 health sciences ,Cas9 ,Transgene ,fungi ,Biomedical Engineering ,food and beverages ,Bioengineering ,Epigenome ,Biology ,Meristem ,Applied Microbiology and Biotechnology ,Cell biology ,03 medical and health sciences ,Tissue culture ,0302 clinical medicine ,Genome editing ,Molecular Medicine ,Guide RNA ,Gene ,030217 neurology & neurosurgery ,030304 developmental biology ,Biotechnology - Abstract
Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
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- 2019
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14. Correction: Building customizable auto-luminescent luciferase-based reporters in plants
- Author
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Nayoung Lee, Furva Rizvi, Colby G. Starker, Ryan Swanson, Arjun Khakhar, Sydney Stokke, Cecily Wang, James C. Chamness, Daniel F. Voytas, and Takato Imaizumi
- Subjects
General Immunology and Microbiology ,QH301-705.5 ,Chemistry ,Science ,General Neuroscience ,Correction ,Plant Biology ,General Medicine ,Plant biology ,General Biochemistry, Genetics and Molecular Biology ,Biochemistry ,Medicine ,Luciferase ,Biology (General) - Published
- 2020
15. Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis
- Author
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Trevor Weiss, Peng Zhou, Maria Elena Gamo, Colby G. Starker, Chunfang Wang, Feng Zhang, Peter A. Crisp, Xiaojun Kang, Daniel F. Voytas, Hui Zhao, and Nathan M. Springer
- Subjects
0301 basic medicine ,0106 biological sciences ,Setaria Plant ,Mutagenesis (molecular biology technique) ,Plant Science ,Computational biology ,Biology ,medicine.disease_cause ,01 natural sciences ,Gene Knockout Techniques ,03 medical and health sciences ,Genome editing ,Genetics ,medicine ,CRISPR ,Multiplex ,Gene ,Plant Proteins ,030304 developmental biology ,Gene Editing ,2. Zero hunger ,0303 health sciences ,Mutation ,Cas9 ,Protoplasts ,Mutagenesis ,Cell Biology ,Plants, Genetically Modified ,Exodeoxyribonucleases ,030104 developmental biology ,CRISPR-Cas Systems ,Functional genomics ,Genome, Plant ,010606 plant biology & botany - Abstract
In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed CRISPR/Cas9 system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples with 94% of deletions larger than 10bp, and less than 1% of mutations being insertions. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical NHEJ DNA repair pathway. Combined with the robust agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at the frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable MMEJ-mediated mutations in many plant species.
- Published
- 2020
- Full Text
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16. Author response: Building customizable auto-luminescent luciferase-based reporters in plants
- Author
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Colby G. Starker, Arjun Khakhar, Furva Rizvi, Sydney Stokke, Cecily Wang, Ryan Swanson, Daniel F. Voytas, James C. Chamness, Takato Imaizumi, and Nayoung Lee
- Subjects
Chemistry ,Luciferase ,Nanotechnology - Published
- 2020
- Full Text
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17. Correction: Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats
- Author
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Renata Peterkova, Zsuzsanna Izsvák, Vladimír Landa, Vladimír Křen, Miroslava Šimáková, Michal Pravenec, Colby G. Starker, Daniel F. Voytas, František Liška, Jan Šilhavý, Petr Mlejnek, Miroslav Peterka, and Vaclav Zidek
- Subjects
Male ,Tail ,Heterozygote ,Genotype ,Science ,Quantitative Trait Loci ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Rats, Inbred SHR ,Transcription Activator-Like Effector Nucleases ,medicine ,Animals ,Abnormalities, Multiple ,Promyelocytic Leukemia Zinc Finger Protein ,Amino Acid Sequence ,Frameshift Mutation ,Gene ,Alleles ,030304 developmental biology ,0303 health sciences ,Transcription activator-like effector nuclease ,Multidisciplinary ,Caudal regression syndrome ,Base Sequence ,Homozygote ,Correction ,Exons ,medicine.disease ,Molecular biology ,Rats ,DNA-Binding Proteins ,Polydactyly ,Gene Targeting ,Medicine ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.
- Published
- 2020
18. Essential nucleotide- and protein-dependent functions of Actb /β-actin
- Author
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Benjamin J. Perrin, Lauren J. Sundby, Angus Lindsay, James M. Ervasti, Pallabi Roy, Colby G. Starker, Daniel F. Voytas, Christopher M. Chamberlain, and Xiaobai Patrinostro
- Subjects
0301 basic medicine ,Multidisciplinary ,ACTG1 ,Cell ,macromolecular substances ,Biology ,Phenotype ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Cytoplasm ,Knockout mouse ,medicine ,Gene ,Actin ,Gene knockout - Abstract
The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.
- Published
- 2018
- Full Text
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19. CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing ofGlycine maxandMedicago truncatula
- Author
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Colby G. Starker, Yer Xiong, Adrian O. Stec, Benjamin W. Campbell, Jean Michel Michno, Andrew L. Eamens, Shaun J. Curtin, Daniel F. Voytas, Tomas Cermak, and Robert M. Stupar
- Subjects
0106 biological sciences ,0301 basic medicine ,Small RNA ,Mutant ,Mutagenesis (molecular biology technique) ,Plant Science ,Biology ,01 natural sciences ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Transcription Activator-Like Effector Nucleases ,Medicago truncatula ,Medicago ,CRISPR ,small RNA ,CRISPR/Cas9 ,Gene ,Research Articles ,soya bean ,Genetics ,CRISPR interference ,Transcription activator-like effector nuclease ,Base Sequence ,Cas9 ,fungi ,RNA-Binding Proteins ,food and beverages ,030104 developmental biology ,Drb2 ,Mutagenesis, Site-Directed ,RNA ,Soybeans ,CRISPR-Cas Systems ,Agronomy and Crop Science ,mutagenesis ,Research Article ,010606 plant biology & botany ,Biotechnology - Abstract
Summary Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soybean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double-mutant for the two soybean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soybean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soybean Dicer-like3 gene and the GmHen1a gene were observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole genome sequencing to reveal a spectrum of non-germline targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops. This article is protected by copyright. All rights reserved.
- Published
- 2017
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20. Downregulation of Plzf Gene Ameliorates Metabolic and Cardiac Traits in the Spontaneously Hypertensive Rat
- Author
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V. Škop, Vladimír Landa, Vladimír Křen, Vaclav Zidek, Massimiliano Mancini, Jaroslava Trnovska, Zsuzsanna Izsvák, Michal Pravenec, Colby G. Starker, Jan Šilhavý, Ondřej Šeda, Petr Mlejnek, Miroslava Šimáková, Ludmila Kazdova, Daniel F. Voytas, František Liška, and Hynek Strnad
- Subjects
0301 basic medicine ,medicine.medical_specialty ,030204 cardiovascular system & hematology ,Biology ,medicine.disease ,Essential hypertension ,Left ventricular hypertrophy ,Transcriptome ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Spontaneously hypertensive rat ,Endocrinology ,Downregulation and upregulation ,Fibrosis ,Internal medicine ,Internal Medicine ,medicine ,Myocardial fibrosis ,Gene - Abstract
The spontaneously hypertensive rat (SHR), one of the most widely used model of essential hypertension, is predisposed to left ventricular hypertrophy, myocardial fibrosis, and metabolic disturbances. Recently, quantitative trait loci influencing blood pressure, left ventricular mass, and heart interstitial fibrosis were genetically isolated within a minimal congenic subline that contains only 7 genes, including mutant Plzf (promyelocytic leukemia zinc finger) candidate gene. To identify Plzf as a quantitative trait gene, we targeted Plzf in the SHR using the transcription activator-like effector nuclease technique and obtained SHR line harboring targeted Plzf gene with a premature stop codon. Because the Plzf targeted allele is semilethal, morphologically normal heterozygous rats were used for metabolic and hemodynamic analyses. SHR- Plzf +/− heterozygotes versus SHR wild-type controls exhibited reduced body weight and relative weight of epididymal fat, lower serum and liver triglycerides and cholesterol, and better glucose tolerance. In addition, SHR- Plzf +/− rats exhibited significantly increased sensitivity of adipose and muscle tissue to insulin action when compared with wild-type controls. Blood pressure was comparable in SHR versus SHR- Plzf +/− ; however, there was significant amelioration of cardiomyocyte hypertrophy and cardiac fibrosis in SHR- Plzf +/− rats. Gene expression profiles in the liver and expression of selected genes in the heart revealed differentially expressed genes that play a role in metabolic pathways, PPAR (peroxisome proliferator-activated receptor) signaling, and cell cycle regulation. These results provide evidence for an important role of Plzf in regulation of metabolic and cardiac traits in the rat and suggest a cross talk between cell cycle regulators, metabolism, cardiac hypertrophy, and fibrosis.
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- 2017
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21. Building customizable auto-luminescent luciferase-based reporters in plants
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Daniel F. Voytas, Ryan Swanson, Cecily Wang, Colby G. Starker, Sydney Stokke, Nayoung Lee, James C. Chamness, Furva Rizvi, Takato Imaizumi, and Arjun Khakhar
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0301 basic medicine ,0106 biological sciences ,plant synthetic biology ,QH301-705.5 ,Computer science ,Science ,Plant Biology ,Computational biology ,01 natural sciences ,Tissue penetration ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Nicotiana benthamiana ,Bioluminescence ,Luciferase ,Biology (General) ,Plant traits ,hormone reporters ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,fungi ,food and beverages ,General Medicine ,luciferase ,Plant biology ,bioluminescence ,Tools and Resources ,Gene expression profiling ,030104 developmental biology ,A. thaliana ,Light driven ,Medicine ,expression reporter ,030217 neurology & neurosurgery ,010606 plant biology & botany - Abstract
Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors., eLife digest Many animals have evolved the capacity to produce light from chemical reactions. For example, an enzyme known as luciferase in fireflies produces light by acting on a molecule called luciferin. Scientists have identified the enzymes that drive several of these systems and used them to build reporters that can study the activity of genes in the tissues of plants and other lifeforms over space and time. However, these reporters often require chemicals to be added to the tissues to produce light. These chemicals tend to be expensive and may not penetrate evenly into the tissues of interest, limiting the potential applications of the reporters in research studies. Recently, it has been discovered that fungi have a bioluminescence pathway that converts a molecule known as caffeic acid into luciferin. Caffeic acid is a common molecule in plants, therefore, it is possible the fungal bioluminescence pathway could be used to build reporters that produce light without needing the addition of chemicals. Now, Khakhar et al. have inserted the genes that encode the enzymes of the fungal bioluminescence pathway into tobacco plants. The experiments found that this was sufficient to turn caffeic acid into molecules of luciferin which are able to produce light. Inserting the same genes into several other plant species, including tomatoes and dahlias, produced similar results. Further experiments showed that the fungal bioluminescence pathway can be used to build reporters that monitor the activity of plant genes throughout living tissues and over a period of several days as well as examine the response to plant hormones. Alongside studying the activities of genes in plants, Khakhar et al. propose that the toolkit developed in this work could be used to generate plants with luminescence that can be switched on or off as desired. This could have many uses including helping plants attract insects to pollinate flowers and building plant biosensors that emit light in response to environmental signals.
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- 2019
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22. Plant gene editing through de novo induction of meristems
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Michael F, Maher, Ryan A, Nasti, Macy, Vollbrecht, Colby G, Starker, Matthew D, Clark, and Daniel F, Voytas
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Gene Editing ,Soil ,Base Sequence ,Seedlings ,CRISPR-Associated Protein 9 ,Meristem ,Mutation ,Tobacco ,Transgenes ,Plants, Genetically Modified ,Plant Shoots ,Plant Proteins - Abstract
Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
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- 2019
23. Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts
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Satya Swathi Nadakuduti, Colby G. Starker, Dae Kwan Ko, Thilani B. Jayakody, C. Robin Buell, Daniel F. Voytas, and David S. Douches
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0106 biological sciences ,0301 basic medicine ,Mutagenesis (molecular biology technique) ,Context (language use) ,Plant Science ,Computational biology ,Biology ,lcsh:Plant culture ,01 natural sciences ,03 medical and health sciences ,double-stranded oligodeoxynucleotides ,Genome editing ,protoplasts ,CRISPR ,lcsh:SB1-1110 ,genome-editing ,CRISPR/Cas9 ,NHEJ ,Original Research ,Transcription activator-like effector nuclease ,Cas9 ,Restriction enzyme ,TALENs ,030104 developmental biology ,Agarose gel electrophoresis ,010606 plant biology & botany - Abstract
Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed.
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- 2019
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24. Genome Editing in Potato with CRISPR/Cas9
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Satya Swathi, Nadakuduti, Colby G, Starker, Daniel F, Voytas, C Robin, Buell, and David S, Douches
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Gene Editing ,Tissue Culture Techniques ,Transformation, Genetic ,Agrobacterium ,CRISPR-Cas Systems ,RNA, Guide, Kinetoplastida ,Solanum tuberosum - Abstract
Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.
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- 2019
25. Genome Editing for Crop Improvement – Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.)
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Satya Swathi Nadakuduti, C. Robin Buell, Daniel F. Voytas, Colby G. Starker, and David S. Douches
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0301 basic medicine ,potato (Solanum tuberosum) ,Plant Science ,lcsh:Plant culture ,Biology ,Crop ,03 medical and health sciences ,Polyploid ,media_common.cataloged_instance ,lcsh:SB1-1110 ,genome-editing ,CRISPR/Cas system ,European union ,polyploidy ,media_common ,Transcription activator-like effector nuclease ,Food security ,Cas9 ,business.industry ,fungi ,food and beverages ,Solanum tuberosum ,Biotechnology ,clonal propagation ,TALENs ,030104 developmental biology ,Agriculture ,business - Abstract
Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.
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- 2018
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26. Essential nucleotide- and protein-dependent functions of
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Xiaobai, Patrinostro, Pallabi, Roy, Angus, Lindsay, Christopher M, Chamberlain, Lauren J, Sundby, Colby G, Starker, Daniel F, Voytas, James M, Ervasti, and Benjamin J, Perrin
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Mice, Knockout ,Cytoplasm ,Mice ,Animals ,macromolecular substances ,Fibroblasts ,Biological Sciences ,Embryo, Mammalian ,Models, Biological ,Actins ,Cell Line - Abstract
Actin is required for many general and specialized cellular functions. Two isoforms, β-actin and γ-actin, are ubiquitously expressed and 99% identical in amino acid sequence, yet previous studies indicated only β-actin is indispensable for life. The nucleotide sequence of each gene also varies, providing additional regulation that may make β-actin indispensable. To separate the effects of protein and nucleotide sequences, the β-actin gene was edited to encode γ-actin protein, while retaining any regulation contained in the nucleotide sequence. The mice, which lack β-actin protein, are viable and appear to be normal. However, they develop progressive hearing loss as auditory sensory cells degenerate. Together, these results show that β-actin function depends on both its nucleotide and protein sequence.
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- 2018
27. Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana
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Colby G. Starker, Daniel F. Voytas, Li Yang, Tieqiang Hu, Jia He, R. Scott Poethig, Kevin McCormick, Matthew R. Willmann, Blake C. Meyers, and Mingli Xu
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0301 basic medicine ,Leaves ,Cancer Research ,Arabidopsis ,Plant Science ,Biochemistry ,Vegetative phase change ,Sequencing techniques ,Gene Expression Regulation, Plant ,Gene expression ,Genetics (clinical) ,Regulation of gene expression ,biology ,Plant Anatomy ,Eukaryota ,Gene Expression Regulation, Developmental ,RNA sequencing ,Trichomes ,Plants ,Plants, Genetically Modified ,Phenotype ,Cell biology ,Nucleic acids ,Phenotypes ,Argonaute Proteins ,Shoot ,Research Article ,lcsh:QH426-470 ,Molecular Probe Techniques ,Research and Analysis Methods ,03 medical and health sciences ,Genetics ,Non-coding RNA ,Molecular Biology Techniques ,Molecular Biology ,Gene ,Psychological repression ,Ecology, Evolution, Behavior and Systematics ,Arabidopsis Proteins ,Gene Expression Profiling ,Organisms ,Biology and Life Sciences ,biology.organism_classification ,Probe Hybridization ,Gene regulation ,Plant Leaves ,MicroRNAs ,lcsh:Genetics ,030104 developmental biology ,Seedlings ,Mutation ,Trans-Activators ,RNA ,sense organs - Abstract
Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression., Author summary Leaves produced at different stages in the development of an Arabidopsis shoot vary predictably in shape and size. Previous studies have shown that this phenomenon is regulated by variation in the abundance of the miRNAs, miR156 and miR157, but how miR156/miR157 produce the changes in leaf morphology that occur during shoot development is not understood. To answer this question, we measured the abundance of miR156/miR157 and their SPL targets in successive leaf primordia, and characterized the effect of variation in the abundance of miR156/miR157 on leaf morphology and the abundance of SPL transcripts and SPL proteins. miR156/miR157 are present at very high levels in the first two rosette leaves, where they act as buffers to stabilize leaf identity. They are present at lower and steadily declining levels in subsequent leaves, where they act to modulate leaf morphogenesis. In these later-formed leaves, a small decrease in the abundance of miR156/miR157 produces a disproportionately large increase in SPL activity, primarily as a result of the increased translation of SPL transcripts. Our results provide a new view of vegetative phase change in Arabidopsis and the mechanism by which miR156 and miR157 regulate this process.
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- 2018
28. Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs
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Lise Christina Deleuran, Daniel F. Voytas, Toni Wendt, Colby G. Starker, Henrik Brinch-Pedersen, Javier Gil-Humanes, and Inger Bæksted Holme
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0106 biological sciences ,0301 basic medicine ,DNA, Bacterial ,Mutant ,Genetic Vectors ,Germination ,Plant Science ,Biology ,01 natural sciences ,03 medical and health sciences ,Mature grain phytase activity ,Oxygen Consumption ,Barley ,Transcription Activator-Like Effector Nucleases ,Gene duplication ,Gene expression ,Genetics ,GCN4/Skn1/RY motif ,Gene ,CRISPR/Cas9 ,6-Phytase ,Base Sequence ,Homozygote ,Wild type ,food and beverages ,HvPAPhy_a ,Promoter ,Hordeum ,General Medicine ,TALENs ,030104 developmental biology ,Mutation ,Seeds ,Phytase ,Hordeum vulgare ,CRISPR-Cas Systems ,Agronomy and Crop Science ,Sequence Alignment ,010606 plant biology & botany - Abstract
In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA. Additionally, the analysis of the HvPAPhy_a promoter region containing the GCN4/Skn1/RY motif highlights its importance for HvPAPhy_a expression as the MGPA in grains of plant lines with mutations within this motif is significantly reduced. Interestingly, lines with deletions located downstream of the motif show even lower MGPA levels, indicating that the GCN4/SKn1/RY motif is not the only element responsible for the level of PAPhy_a expression during grain maturation. Mutant grains with very low MPGA showed delayed germination as compared to grains of wild type barley. As grains with high levels of preformed phytases would provide more readily available phosphorous needed for a fast germination, this indicates that faster germination may be implicated in the positive selection of the ancient PAPhy gene duplication that lead to the creation of the PAPhy_a gene.
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- 2017
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29. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9
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Susana Sánchez-León, Nicholas J. Baltes, Daniel F. Voytas, Colby G. Starker, Yanpeng Wang, Qiwei Shan, Javier Gil-Humanes, Zhen Liang, Francisco Barro, Carmen V. Ozuna, Caixia Gao, National Science Foundation (US), National Natural Science Foundation of China, and Fundación Alfonso Martín Escudero
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0301 basic medicine ,DNA, Bacterial ,Agrobacterium ,Plant Science ,Genome ,Article ,DNA replicons ,Genome engineering ,03 medical and health sciences ,Genome editing ,Solanum lycopersicum ,Tobacco ,Genetics ,CRISPR ,Homologous recombination ,Technical advance ,CRISPR/Cas9 ,Triticum ,Gene Editing ,biology ,Cas9 ,fungi ,Gene targeting ,food and beverages ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,Gene Targeting ,Wheat ,Replicon ,CRISPR-Cas Systems ,Multiplexed gene targeting - Abstract
Gil Humanes, Javier et al., The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome., This work was supported in part by grants to D.F.V. from the National Science Foundation (IOS-1444511and IOS-1339209), and to C.G. from the National Natural Science Foundation of China (31420103912). Javier Gil-Humanes acknowledges the Fundación Alfonso Martin Escudero for his post-doctoral fellowship.
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- 2017
30. Targeted Mutagenesis of the Tomato PROCERA Gene Using Transcription Activator-Like Effector Nucleases
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David Weiss, Vai S. Lor, Daniel F. Voytas, Colby G. Starker, and Neil E. Olszewski
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Crops, Agricultural ,Transcription activator-like effector nuclease ,Base Sequence ,Physiology ,Effector ,Molecular Sequence Data ,fungi ,Mutant ,food and beverages ,Plant Science ,Biology ,Endonucleases ,Cell biology ,Solanum lycopersicum ,Mutagenesis ,Transcription (biology) ,Botany ,Genetics ,Base sequence ,Gene ,Scientific Correspondence ,Plant Proteins - Abstract
Transcription activator-like effector nucleases successfully generate a heritable tomato mutant.
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- 2014
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31. TALEN-based Gene Correction for Epidermolysis Bullosa
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Anthony P. DeFeo, Richard Gabriel, Amber N. McElroy, Megan J. Riddle, John E. Wagner, Manfred Schmidt, Morgan L. Maeder, J. Keith Joung, Christof von Kalle, Bruce R. Blazar, Daniel F. Voytas, Daniel F. Carlson, Beau R. Webber, Jakub Tolar, Colby G. Starker, Lily Xia, and Mark J. Osborn
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Male ,Transcriptional Activation ,Collagen Type VII ,Genotype ,Induced Pluripotent Stem Cells ,Molecular Sequence Data ,Genes, Recessive ,Gene mutation ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Drug Discovery ,Genetics ,COL7A1 Gene ,medicine ,Humans ,DNA Breaks, Double-Stranded ,Selection, Genetic ,Molecular Biology ,Gene ,030304 developmental biology ,Pharmacology ,Base Composition ,0303 health sciences ,Transcription activator-like effector nuclease ,Deoxyribonucleases ,Gene Transfer Techniques ,Epidermolysis bullosa dystrophica ,Chromosome Mapping ,High-Throughput Nucleotide Sequencing ,Recombinational DNA Repair ,Reproducibility of Results ,Gene targeting ,Genetic Therapy ,Fibroblasts ,medicine.disease ,Molecular biology ,Epidermolysis Bullosa Dystrophica ,HEK293 Cells ,Phenotype ,Genetic Loci ,030220 oncology & carcinogenesis ,Gene Targeting ,Molecular Medicine ,Original Article ,Epidermolysis bullosa ,Gene Deletion - Abstract
Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.
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- 2013
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32. Downregulation of
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František, Liška, Vladimír, Landa, Václav, Zídek, Petr, Mlejnek, Jan, Šilhavý, Miroslava, Šimáková, Hynek, Strnad, Jaroslava, Trnovská, Vojtěch, Škop, Ludmila, Kazdová, Colby G, Starker, Daniel F, Voytas, Zsuzsanna, Izsvák, Massimiliano, Mancini, Ondřej, Šeda, Vladimír, Křen, and Michal, Pravenec
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Male ,Analysis of Variance ,Gene Expression Profiling ,Blotting, Western ,Quantitative Trait Loci ,Kruppel-Like Transcription Factors ,Down-Regulation ,Blood Pressure Determination ,Lipid Metabolism ,Real-Time Polymerase Chain Reaction ,Fibrosis ,Rats ,Phenotype ,Rats, Inbred SHR ,Hypertension ,Animals ,Hypertrophy, Left Ventricular ,Myocytes, Cardiac ,Promyelocytic Leukemia Zinc Finger Protein ,Essential Hypertension ,Alleles ,Cells, Cultured - Abstract
The spontaneously hypertensive rat (SHR), one of the most widely used model of essential hypertension, is predisposed to left ventricular hypertrophy, myocardial fibrosis, and metabolic disturbances. Recently, quantitative trait loci influencing blood pressure, left ventricular mass, and heart interstitial fibrosis were genetically isolated within a minimal congenic subline that contains only 7 genes, including mutant
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- 2016
33. Targeting of the Plzf gene in the rat by transcription activator-like effector nuclease results in caudal regression syndrome in spontaneously hypertensive rats
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Daniel F. Voytas, František Liška, Petr Mlejnek, Vladimír Landa, Michal Pravenec, Miroslav Peterka, Colby G. Starker, Zsuzsanna Izsvák, Vaclav Zidek, Renata Peterkova, Jan Šilhavý, Miroslava Šimáková, and Vladimír Křen
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0301 basic medicine ,Candidate gene ,Embryology ,Vertebrae ,Physiology ,Science ,Biology ,Frameshift mutation ,Pelvis ,03 medical and health sciences ,Exon ,Spontaneously hypertensive rat ,medicine ,Medicine and Health Sciences ,Allele ,Animal Anatomy ,Gene ,Musculoskeletal System ,Genetics ,Tails ,Fetuses ,Multidisciplinary ,Caudal regression syndrome ,Body Weight ,Gene targeting ,Biology and Life Sciences ,Kidneys ,Renal System ,medicine.disease ,Molecular biology ,Spine ,030104 developmental biology ,Physiological Parameters ,Cardiovascular and Metabolic Diseases ,Medicine ,Anatomy ,Ureter ,Zoology ,Research Article ,Developmental Biology - Abstract
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.
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- 2016
34. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14
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Daniel F. Voytas, Colby G. Starker, Richard Seonghun Nho, Do Hyung Kim, Douglas Grunwald, Ji Man Park, Chang Hwa Jung, Neil Michael Otto, Branden S. Moriarity, Young Mi Kim, Minchul Seo, and Kwan Hyun Kim
- Subjects
0301 basic medicine ,Class I Phosphatidylinositol 3-Kinases ,Vesicular Transport Proteins ,Autophagy-Related Proteins ,mTORC1 ,Biology ,Mechanistic Target of Rapamycin Complex 1 ,Cell Line ,03 medical and health sciences ,Mice ,Phosphoserine ,0302 clinical medicine ,Autophagy ,Animals ,Autophagy-Related Protein-1 Homolog ,Humans ,Amino Acid Sequence ,Kinase activity ,Phosphorylation ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Phosphatidylethanolamines ,TOR Serine-Threonine Kinases ,Autophagosomes ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,BECN1 ,Autophagy-related protein 13 ,Basic Research Paper ,Cell biology ,Up-Regulation ,Adaptor Proteins, Vesicular Transport ,030104 developmental biology ,030220 oncology & carcinogenesis ,Multiprotein Complexes ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.
- Published
- 2016
35. Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration
- Author
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Andressa Mota, Robert D. Goldman, Karlyndsay Sitterley, Liya Ding, Jason Lowery, Nancy Costigliola, Christoph J. Burckhardt, Assaf Zaritsky, Jessica D. Tytell, Zhuo Gan, Gaudenz Danuser, Daniel F. Voytas, and Colby G. Starker
- Subjects
0301 basic medicine ,Histology ,Polarity (physics) ,Microtubule-associated protein ,viruses ,Cell ,Intermediate Filaments ,Vimentin ,Biology ,Microtubules ,Article ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Cell Movement ,Tubulin ,Cell polarity ,medicine ,Cytoskeleton ,Intermediate filament ,Cells, Cultured ,virus diseases ,Cell Polarity ,Cell migration ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Cell biology ,medicine.anatomical_structure ,030104 developmental biology ,biology.protein ,Persistence (discontinuity) ,Microtubule-Associated Proteins ,030217 neurology & neurosurgery - Abstract
Increased expression of vimentin intermediate filaments (VIFs) enhances directed cell migration, but the mechanism behind VIFs' effect on motility is not understood. VIFs interact with microtubules, whose organization contributes to polarity maintenance in migrating cells. Here, we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of retinal pigment epithelial cells. By genome editing, we fluorescently labeled endogenous vimentin and α-tubulin, and we developed computational image analysis to delineate architecture and interactions of the two networks. Our results show that VIFs assemble an ultrastructural copy of the previously polarized microtubule network. Because the VIF network is long-lived compared to the microtubule network, VIFs template future microtubule growth along previous microtubule tracks, thus providing a feedback mechanism that maintains cell polarity. VIF knockdown prevents cells from polarizing and migrating properly during wound healing. We suggest that VIFs' templating function establishes a memory in microtubule organization that enhances persistence in cell polarization in general and migration in particular.
- Published
- 2016
36. In vivo genome editing using a high-efficiency TALEN system
- Author
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Alvin C.H. Ma, Scott C. Fahrenkrug, Stephen C. Ekker, Sumedha G. Penheiter, Daniel F. Carlson, Anskar Y.H. Leung, Randall G. Krug, Jarryd M. Campbell, Karl J. Clark, Jeffrey J. Essner, Victoria M. Bedell, Daniel F. Voytas, Ying Wang, Tanya L. Poshusta, Wenfang Tan, and Colby G. Starker
- Subjects
Genetics ,0303 health sciences ,Transcription activator-like effector nuclease ,Multidisciplinary ,Morpholino ,biology ,Genomics ,biology.organism_classification ,Genome ,Genome engineering ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Gene ,Zebrafish ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
- Published
- 2012
- Full Text
- View/download PDF
37. Conservation in Function of a SCAR/WAVE Component During Infection Thread and Root Hair Growth in Medicago truncatula
- Author
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Lucinda S. Smith, Giles E. D. Oldroyd, Jennifer H. Richens, Colby G. Starker, Akira Miyahara, Giulia Morieri, Sharon R. Long, and J. Allan Downie
- Subjects
Regulation of gene expression ,biology ,Physiology ,Molecular Sequence Data ,fungi ,food and beverages ,Locus (genetics) ,General Medicine ,Root hair ,biology.organism_classification ,Plant Root Nodulation ,Plant Roots ,Medicago truncatula ,Trichome ,Cell biology ,Gene Expression Regulation, Plant ,Mutation ,Gene expression ,Botany ,Symbiosis ,Agronomy and Crop Science ,Gene ,Actin ,Plant Proteins - Abstract
Nitrogen-fixing symbioses of plants are often associated with bacterially infected nodules where nitrogen fixation occurs. The plant host facilitates bacterial infection with the formation of infection threads, unique structures associated with these symbioses, which are invaginations of the host cell with the capability of traversing cellular junctions. Here, we show that the infection thread shares mechanistic similarities to polar-growing cells, because the required for infection thread (RIT) locus of Medicago truncatula has roles in root-hair, trichome, and infection-thread growth. We show that RIT encodes the M. truncatula ortholog of NAP1, a component of the SCAR/WAVE (suppressor of cAMP receptor/WASP-family verprolin homologous protein) complex that regulates actin polymerization, through the activation of ARP2/3. NAP1 of Arabidopsis thaliana functions equivalently to the M. truncatula gene, indicating that the mode of action of NAP1 is functionally conserved across species and that legumes have not evolved a unique functionality for NAP1 during rhizobial colonization. This work highlights the surprising commonality between polar-growing cells and a polar-growing cellular intrusion and reveals important insights into the formation and maintenance of infection-thread development.
- Published
- 2010
- Full Text
- View/download PDF
38. An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction
- Author
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John F. Marsh, Sharon R. Long, György B. Kiss, Raka M. Mitra, Brigitta Dudas, Júlia Jakab, Kathryn A. VandenBosch, Radhika Prabhu, Colby G. Starker, Jake Doll, Doug R. Cook, Giles E. D. Oldroyd, Péter Kaló, Patrick H. Middleton, R. Varma Penmetsa, and Attila Kereszt
- Subjects
Lipopolysaccharides ,Genetics ,Sequence Homology, Amino Acid ,Transcription, Genetic ,Molecular Sequence Data ,Plant Root Nodulation ,food and beverages ,Cell Biology ,Plant Science ,DNA-binding domain ,Nod ,Biology ,Nod factor ,Gene Expression Regulation, Plant ,Gene expression ,Medicago ,Amino Acid Sequence ,Signal transduction ,Sequence Alignment ,Gene ,Transcription factor ,Research Articles ,Plant Proteins ,Signal Transduction ,Transcription Factors - Abstract
Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.
- Published
- 2007
- Full Text
- View/download PDF
39. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice
- Author
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Tomas Cermak, Seiichi Toki, Daniel F. Voytas, Hiroaki Saika, Colby G. Starker, Akiko Mori, Ayako Nishizawa-Yokoi, Keishi Osakabe, Kazuhiko Sugimoto, Masao Hamada, Yuichi Katayose, and Tomoki Hoshino
- Subjects
0301 basic medicine ,DNA End-Joining Repair ,DNA Ligases ,DNA, Plant ,Physiology ,Mutant ,Plant Science ,LIG4 ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Transcription Activator-Like Effector Nucleases ,Genetics ,DNA Breaks, Double-Stranded ,Gene ,Plant Proteins ,Transcription activator-like effector nuclease ,urogenital system ,fungi ,Wild type ,Oryza ,Breakthrough Technologies ,Plants, Genetically Modified ,Non-homologous end joining ,030104 developmental biology ,chemistry ,Mutation ,Mutagenesis, Site-Directed ,DNA - Abstract
We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.
- Published
- 2015
40. Fanconi Anemia Gene Editing by the CRISPR/Cas9 System
- Author
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Christof von Kalle, J. Keith Joung, Jordan Jarjour, Richard Gabriel, Amber N. McElroy, Bruce R. Blazar, Daniel F. Voytas, Colby G. Starker, Mark J. Osborn, John E. Wagner, Manfred Schmidt, Anthony P. DeFeo, Jakub Tolar, and Beau R. Webber
- Subjects
Streptococcus pyogenes ,Molecular Sequence Data ,Primary Cell Culture ,Gene Expression ,Biology ,Gene mutation ,Transfection ,Genome ,Polymerase Chain Reaction ,Genome engineering ,Genome editing ,Bacterial Proteins ,Genetics ,CRISPR ,Deoxyribonuclease I ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Molecular Targeted Therapy ,Molecular Biology ,Gene ,Research Articles ,Base Sequence ,Cas9 ,Genome, Human ,Fanconi Anemia Complementation Group C Protein ,Fibroblasts ,Lipids ,Electroporation ,Fanconi Anemia ,Genetic Loci ,Molecular Medicine ,Human genome ,Genetic Engineering ,Plasmids ,RNA, Guide, Kinetoplastida - Abstract
Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
- Published
- 2014
41. Efficient design and assembly of custom TALENs using the Golden Gate platform
- Author
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Tomas, Cermak, Colby G, Starker, and Daniel F, Voytas
- Subjects
Homeodomain Proteins ,Gene Targeting ,Genetic Vectors ,Protein Interaction Domains and Motifs ,Cloning, Molecular ,Endonucleases ,Genetic Engineering ,Homologous Recombination ,Gene Library - Abstract
An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other sequence-specific nucleases. In this chapter, we describe a rapid, inexpensive, and user-friendly protocol for custom TALEN construction based on one of the most popular TALEN assembly platforms, the Golden Gate cloning method. Using this protocol, ready-to-use TALENs with specificity for targets 13-32 bp long are constructed within 5 days.
- Published
- 2014
42. Efficient Design and Assembly of Custom TALENs Using the Golden Gate Platform
- Author
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Daniel F. Voytas, Tomas Cermak, and Colby G. Starker
- Subjects
Genetics ,Cloning ,Transcription activator-like effector nuclease ,Effector ,Computer science ,Golden Gate Cloning ,Gene targeting ,Locus (genetics) ,Computational biology ,DNA-binding domain ,DNA sequencing ,Genome engineering ,chemistry.chemical_compound ,chemistry ,Transcription (biology) ,Genomic library ,Homologous recombination ,Gene ,DNA ,Gene knockout - Abstract
An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other sequence-specific nucleases. In this chapter, we describe a rapid, inexpensive, and user-friendly protocol for custom TALEN construction based on one of the most popular TALEN assembly platforms, the Golden Gate cloning method. Using this protocol, ready-to-use TALENs with specificity for targets 13-32 bp long are constructed within 5 days.
- Published
- 2014
- Full Text
- View/download PDF
43. Tailor-made mutations in Arabidopsis using zinc finger nucleases
- Author
-
Yiping, Qi, Colby G, Starker, Feng, Zhang, Nicholas J, Baltes, and Daniel F, Voytas
- Subjects
DNA End-Joining Repair ,Transformation, Genetic ,Base Sequence ,DNA, Plant ,Mutagenesis ,DNA Mutational Analysis ,Molecular Sequence Data ,Arabidopsis ,DNA Breaks, Double-Stranded ,Zinc Fingers ,Cloning, Molecular ,Deoxyribonucleases, Type II Site-Specific ,Plants, Genetically Modified - Abstract
Zinc finger nucleases (ZFNs) are proteins engineered to make site-specific double-strand breaks (DSBs) in a DNA sequence of interest. Imprecise repair of the ZFN-induced DSBs by the nonhomologous end-joining (NHEJ) pathway results in a spectrum of mutations, such as nucleotide substitutions, insertions, and deletions. Here we describe a method for targeted mutagenesis in Arabidopsis with ZFNs, which are engineered by context-dependent assembly (CoDA). This ZFN-induced mutagenesis method is an alternative to other currently available gene knockout or knockdown technologies and is useful for reverse genetic studies.
- Published
- 2013
44. Targeted mutagenesis for functional analysis of gene duplication in legumes
- Author
-
Shaun J, Curtin, Justin E, Anderson, Colby G, Starker, Nicholas J, Baltes, Dhananjay, Mani, Daniel F, Voytas, and Robert M, Stupar
- Subjects
Internet ,Mutagenesis ,Gene Duplication ,Gene Targeting ,Computational Biology ,Fabaceae ,Zinc Fingers ,Soybeans ,Codon ,Endonucleases - Abstract
Assessment of gene function oftentimes requires mutant populations that can be screened by forward or reverse genetic analysis. The situation becomes more complicated in polyploidy or paleopolyploid genomes that have two or more copies for most genes. Here we describe a method for engineering zinc-finger nucleases (ZFNs) for the purpose of creating targeted mutations in the paleopolyploid soybean genome. ZFNs are recombinant proteins composed of an engineered zinc-finger array fused to a nonspecific cleavage domain. When engineered to recognize a specific nucleotide sequence, the cleavage domain will generate highly mutagenic DNA double-strand breaks frequently resulting in insertions and deletions at the target locus. Depending on the number of target sites present within the genome, this method has the capacity to target either single- or multi-copy gene families. In this chapter, we describe an inexpensive, rapid, and user-friendly approach for ZFN assembly and application in soybean based on the previously described context-dependent assembly method.
- Published
- 2013
45. Gene silencing in Medicago truncatula roots using RNAi
- Author
-
Daniela S, Floss, Alexa M, Schmitz, Colby G, Starker, J Stephen, Gantt, and Maria J, Harrison
- Subjects
Gene Expression Regulation, Plant ,Genetic Vectors ,Medicago truncatula ,RNA Interference ,Gene Silencing ,Plants, Genetically Modified ,Symbiosis ,Plant Roots ,Rhizobium - Abstract
Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.
- Published
- 2013
46. Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases
- Author
-
Jeffry D. Sander, Xiaohong Li, Yiping Qi, Daniel F. Voytas, J. Keith Joung, Colby G. Starker, Nicholas J. Baltes, Yong Zhang, Feng Zhang, and Deepak Reyon
- Subjects
0106 biological sciences ,zinc finger nuclease (ZFN) ,Molecular Sequence Data ,Arabidopsis ,Chimeric gene ,Biology ,Investigations ,Genes, Plant ,01 natural sciences ,Genome ,03 medical and health sciences ,inversion ,Gene cluster ,Tandemly arrayed genes ,Genetics ,deletion ,Molecular Biology ,Genetics (clinical) ,030304 developmental biology ,Zinc finger ,0303 health sciences ,Endodeoxyribonucleases ,Base Sequence ,Gene targeting ,Zinc Fingers ,tandemly arrayed genes (TAGs) ,Plants, Genetically Modified ,Zinc finger nuclease ,Tandem Repeat Sequences ,Multigene Family ,Gene Targeting ,Genetic redundancy ,Mutagenesis, Site-Directed ,Chromosome Deletion ,010606 plant biology & botany - Abstract
Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology.
- Published
- 2013
47. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants
- Author
-
Daniel F. Voytas, Toni Wendt, Henrik Brinch-Pedersen, Preben Bach Holm, Inger Bæksted Holme, Colby G. Starker, and Michelle Christian
- Subjects
Genetics ,Transcription activator-like effector nuclease ,Base Sequence ,Effector ,Molecular Sequence Data ,Hordeum ,Plant Science ,General Medicine ,Biology ,Endonucleases ,Zinc finger nuclease ,Article ,Genome engineering ,Transformation, Genetic ,TAL effector ,Genome editing ,Transcription (biology) ,Sequence Homology, Nucleic Acid ,Mutation ,Hordeum vulgare ,Agronomy and Crop Science ,Genome, Plant ,Transcription Factors - Abstract
Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.
- Published
- 2013
- Full Text
- View/download PDF
48. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways
- Author
-
Colby G. Starker, Yungil Ryu, Daniel F. Voytas, Joshua A. Baller, Yiping Qi, Feng Zhang, Spencer C. Cleland, and Yong Zhang
- Subjects
Ku80 ,DNA End-Joining Repair ,DNA Ligases ,DNA Repair ,DNA repair ,Arabidopsis ,Method ,Cell Cycle Proteins ,Biology ,Genome editing ,Genetics ,Homologous Recombination ,Genetics (clinical) ,Transcription activator-like effector nuclease ,Arabidopsis Proteins ,Gene Expression Profiling ,fungi ,Zinc Fingers ,DNA Repair Pathway ,Endonucleases ,Plants, Genetically Modified ,Ku Protein ,DNA-Binding Proteins ,Genetic Loci ,Gene Targeting ,Mutation ,Mutagenesis, Site-Directed ,Mitogen-Activated Protein Kinases ,Homologous recombination ,Genome, Plant - Abstract
Customizable, sequence-specific nucleases make it possible to precisely modify the genomes of many higher organisms, including diverse plant species (Voytas 2013). There are three primary types of sequence-specific nucleases, namely, zinc finger nucleases (ZFNs) (Kim et al. 1996; Carroll 2011), TAL effector nucleases (TALENs) (Christian et al. 2010; Bogdanove and Voytas 2011), and meganucleases (Smith et al. 2006; Paques and Duchateau 2007). All three nuclease types introduce targeted DNA double-strand breaks (DSBs), which activate the cell's DNA repair pathways, principally nonhomologous end-joining (NHEJ) and homologous recombination (HR) (Kanaar et al. 1998; Puchta 2005; Hartlerode and Scully 2009). NHEJ predominates in nonreplicating cells and repair is often imprecise, such that mutations are introduced at the cut site. HR, which enables gene replacement or gene targeting (GT), predominates in replicating cells. Because genome modifications made with customizable nucleases rely on DSB repair, it should be possible to manipulate DNA repair pathways to influence the type and frequency of targeted modifications attained. For example, promoting NHEJ should enhance nuclease-mediated site-specific mutagenesis; however, there are no reports of this approach being successful in plants. Strategies to enhance HR have been reported in a few plant studies; for example, high-frequency HR was obtained by overexpressing the yeast RAD54 gene (Shaked et al. 2005). Enhanced HR was also attained in Arabidopsis by knocking out RAD50 (Gherbi et al. 2001) or CAF1(Endo et al. 2006) or by overexpressing SMC6B (also known as MIM) (Hanin et al. 2000). Almost all of these studies used a GUS transgene reporter that measures intrachromosomal, single-strand annealing (SSA) and/or synthesis-dependent strand annealing (SDSA) in which sister chromatids or homologous chromosomes are used as repair templates (Orel et al. 2003; Puchta 2005). For GT, exogenous donors or chromosomally integrated, nonallelic donors are usually used. The repair mechanisms used in GT, therefore, could be quite different, and it remains to be determined whether previously demonstrated manipulations of DNA repair pathways will apply to GT of endogenous genes. One of the few attempts to influence GT in plants involved overexpression of the bacterial RecA protein, which enhanced the fidelity of DSB-induced GT in tobacco but not the overall GT frequency (Reiss et al. 2000). In this study, we tested the consequence of manipulating key regulators of NHEJ and HR on targeted mutagenesis or GT mediated by sequence-specific nucleases. In classic NHEJ (C-NHEJ), KU70 dimerizes with KU80 to form KU protein complexes, which directly bind to DSBs to initiate repair (Pastwa and Blasiak 2003). In HR, SMC6 acts when sister chromatids are used as repair templates in both human (De Piccoli et al. 2006; Potts et al. 2006) and Arabidopsis cells (Watanabe et al. 2009). SMC6 dimerizes with SMC5 to form SMC6/5 complexes that initiate repair of DSBs using sister chromatids as templates (Potts 2009). The upstream positions of KU70 and SMC6 in the NHEJ or HR pathways, respectively, made them good candidates as regulators of DNA repair pathway choice. We also chose LIG4, because it carries out a well-known and conserved function downstream from KU (Pastwa and Blasiak 2003). A ZFN that targets the endogenous Arabidopsis ADH1 gene was used to create DSBs (Zhang et al. 2010), and NHEJ mutagenesis and GT were measured in whole seedlings and rosette leaf protoplasts in each of the three DNA repair mutant backgrounds. Our results clearly demonstrate that manipulating these key DNA repair genes in Arabidopsis can influence the efficiencies and outcomes of targeted mutagenesis and GT.
- Published
- 2013
49. Targeted Mutagenesis for Functional Analysis of Gene Duplication in Legumes
- Author
-
Colby G. Starker, Robert M. Stupar, Shaun J. Curtin, Justin E. Anderson, Daniel F. Voytas, Dhananjay Mani, and Nicholas J. Baltes
- Subjects
Genetics ,Transcription activator-like effector nuclease ,fungi ,Gene duplication ,Gene family ,Gene targeting ,Locus (genetics) ,Biology ,Gene ,Zinc finger nuclease ,Genome - Abstract
Assessment of gene function oftentimes requires mutant populations that can be screened by forward or reverse genetic analysis. The situation becomes more complicated in polyploidy or paleopolyploid genomes that have two or more copies for most genes. Here we describe a method for engineering zinc-finger nucleases (ZFNs) for the purpose of creating targeted mutations in the paleopolyploid soybean genome. ZFNs are recombinant proteins composed of an engineered zinc-finger array fused to a nonspecific cleavage domain. When engineered to recognize a specific nucleotide sequence, the cleavage domain will generate highly mutagenic DNA double-strand breaks frequently resulting in insertions and deletions at the target locus. Depending on the number of target sites present within the genome, this method has the capacity to target either single- or multi-copy gene families. In this chapter, we describe an inexpensive, rapid, and user-friendly approach for ZFN assembly and application in soybean based on the previously described context-dependent assembly method.
- Published
- 2013
- Full Text
- View/download PDF
50. Gene Silencing in Medicago truncatula Roots Using RNAi
- Author
-
Alexa M. Schmitz, Maria J. Harrison, Daniela S. Floss, J. Stephen Gantt, and Colby G. Starker
- Subjects
Gene knockdown ,Agrobacterium ,fungi ,food and beverages ,Computational biology ,Biology ,biology.organism_classification ,Medicago truncatula ,Transformation (genetics) ,RNA interference ,Botany ,Gene silencing ,Gene ,Functional genomics - Abstract
Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.
- Published
- 2013
- Full Text
- View/download PDF
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