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22 results on '"Cermak, Tomas"'

8. Targeting DNA double-strand breaks with TAL effector nucleases

Author

Christian, Michelle, Cermak, Tomas, Doyle, Erin L., Schmidt, Clarice, Feng Zhang, Hummel, Aaron, Bogdanove, Adam J., and Voytas, Daniel F.

Subjects
DNA sequencing -- Research, Nucleotide sequencing -- Research, Mutagenesis -- Research, Nucleases -- Research, Genetic transcription -- Research, Biological sciences
Published
2010

10. Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Author

Hummel, Aaron W., Chauhan, Raj Deepika, Cermak, Tomas, Mutka, Andrew M., Vijayaraghavan, Anupama, Boyher, Adam, Starker, Colby G., Bart, Rebecca, Voytas, Daniel F., and Taylor, Nigel J.

Subjects
Manihot, gene editing, herbicide tolerance, Herbicides, fungi, Glycine, food and beverages, gene replacement, Genes, Plant, Plants, Genetically Modified, cassava, Genetic Loci, 3-Phosphoshikimate 1-Carboxyvinyltransferase, Genetic Engineering, Research Articles, Alleles, Research Article, Herbicide Resistance
Abstract

Summary Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.

Published
2018

15. Mouse genome engineering using designer nucleases

Author

Hermann, Mario, Cermak, Tomas, Voytas, Daniel F, Pelczar, Pawel, Hermann, Mario, Cermak, Tomas, Voytas, Daniel F, and Pelczar, Pawel

Abstract

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Published
2014

22. Efficient design and assembly of custom TALENs using the Golden Gate platform.

Author

Cermak T, Starker CG, and Voytas DF

Subjects
Cloning, Molecular methods, Endonucleases chemistry, Gene Library, Gene Targeting, Homeodomain Proteins chemistry, Homologous Recombination, Endonucleases genetics, Endonucleases metabolism, Genetic Engineering methods, Genetic Vectors genetics, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Protein Interaction Domains and Motifs
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
2015
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