Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells


Typ / Jahr

Journal Article / 2009

Autoren

Tovkach, Andriy; Zeevi, Vardit; Tzfira, Tzvi

Abstract

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a FokI endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta.

Keywords
gene targeting; non-homologous end joining.; tools; Zinc finger nucleases
Periodical
The Plant journal : for cell and molecular biology
Periodical Number
4
Page range
747–757
Volume
57
DOI
10.1111/j.1365-313X.2008.03718.x

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
497 Tzfira, Tzvi
USA
Arabidopsis thaliana Zinc-finger nucleases
GUS, uidA
GUS expression
SDN1
Basic research
Basic research
498 Tzfira, Tzvi
USA
Nicotiana benthamiana Zinc-finger nucleases
GUS, uidA
GUS expression
SDN3
Basic research
Basic research