TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

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.

Induced Variations in Brassinosteroid Genes Define Barley Height and Sturdiness, and Expand the Green Revolution Genetic Toolkit

“…An alternative approach is to create mutations through targeted mutagenesis and genome editing. However, these techniques are still in their infancy in barley (Wendt et al, 2013) and although it would be possible to create mutations without leaving any traces of foreign DNA in the resulting breeding lines, most countries regard such plants as genetically modified or legal decisions are pending. Regardless of the outcome of such decisions, public opinion has to show a positive acceptance to gene editing before it will be of economic importance.…”

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

“…Moreover, TALENs appear to be more mutagenic than ZFNs (Chen et al, 2013) and are highly specific (Juillerat et al, 2014). To promote TALEN technology, we developed a streamlined TALEN assembly method (Cermak et al, 2011), which was later used for successful genome editing in many plant species Shan et al, 2013a;Wendt et al, 2013;Zhang et al, 2013;Lor et al, 2014;Wang et al, 2014). Unfortunately, both ZFN and TALEN technologies require engineering of DNA-binding domains for individual targeting applications, demanding significant effort and expertise in molecular cloning.…”

Targeted Mutagenesis of the Tomato PROCERA Gene Using Transcription Activator-Like Effector Nucleases

“…Binding of TALEN monomers to the targeted gene allows the FokI nuclease domain to dimerize and cleave the DNA (Chen and Gao, 2013;Voytas, 2013). TALENs have been successfully used to make sequence-specific mutations in a variety of plant species, including Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), rice (Oryza sativa), Brachypodium spp., and barley (Hordeum vulgare; Cermak et al, 2011;Li et al, 2012;Christian et al, 2013;Shan et al, 2013a;Wendt et al, 2013;Zhang et al, 2013). More recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein9 (Cas9) system has also been demonstrated to be a promising genome-editing tool (Jiang et al, 2013;Li et al, 2013;Nekrasov et al, 2013;Shan et al, 2013b;Upadhyay et al, 2013).…”