TALE nucleases: tailored genome engineering made easy

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Custom-made designer nucleases have evolved into an indispensable platform to precisely alter complex genomes for basic research, biotechnology, synthetic biology, or human gene therapy. In this review we describe how transcription activator-like effector nucleases (TALENs) have rapidly developed into a chief technology for targeted genome editing in different model organisms as well as human stem cells. We summarize the technological background and provide an overview of the current state-of-the-art of TALENs with regard to activity and specificity of these nucleases for targeted genome engineering.

Graphical abstract

TALEN-mediated genome engineering. After a TALEN pair has cleaved the target locus, the resulting DNA double strand break will be repaired by either homologous recombination (HR)-based repair or non-homologous end-joining (NHEJ). In the absence of a repair template, the broken chromosome is re-ligated by the error-prone NHEJ pathway. The resulting small insertions and deletions (asterisk) will disrupt the open reading frame. Targeted deletions can be obtained by expressing two TALEN pairs that cut two adjacent sites on a chromosome. In the presence of donor DNA, the homologous sequences can either be employed to correct a mutation (asterisk) in the genome or to target integration of a transgene into a chosen site.

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Highlights

► Design of customized transcription activator-like effector nucleases (TALENs). ► Potential of TALENs in genome editing applications. ► Comparison to other designer nuclease platforms, such as zinc-finger nucleases.

Introduction

The ability to precisely manipulate complex genomes has constituted a significant leap forward for researchers interested in basic and applied genetics. Since the first methods to generate knockout and knockin mouse models were developed in the late 1980s [1], scientists could selectively modify a gene to explore its function in vivo. About two decades later, the generation of customized nucleases enabled reverse genetics to spread beyond the well-known model organisms [2, 3]. Ever since the discovery that the creation of a targeted DNA double-strand break (DSB) enhances the frequency of homologous recombination (HR)-mediated repair at the targeted locus [4, 5], great effort has been invested in improving the design and the efficacy of tailored nucleases. Traditionally, zinc-finger nucleases (ZFNs) [6] and meganucleases [7, 8] have been used for that purpose but more recently designer nucleases based on transcriptional activator-like effectors (TALEs) have been successfully applied in a variety of organisms [9•, 10•, 11•, 12•] and stem cells [13•]. Here, we review the development of customized TALE nucleases (TALENs) and their potential for genome engineering (see graphical abstract), including applications in basic research, systems biology, biotechnology, human gene therapy, and highlight the advantages and limitations of this novel genome editing platform.

Section snippets

A novel DNA binding domain

TALEs are bacterial proteins that are injected into infected plant cells via a type III secretion system by pathogens of the genus Xanthomonas [14, 15]. These proteins are typically composed of a N-terminal translocation domain, a central repeat domain that mediates binding to the DNA, and a C-terminal transcriptional activation domain (Figure 1). Once in the cell, these factors recognize their DNA targets in the host genome and activate the expression of genes necessary for pathogen

Targeted genome engineering

Many studies performed in the last five years have demonstrated the great potential of designer nucleases in targeted genome engineering [3, 6, 25, 26•]. These artificial enzymes are generally composed of a customized DNA binding domain fused to the non-specific DNA cleavage domain of the FokI restriction enzyme [27]. Thus far, ZFNs have been the most commonly used platform to precisely modify the genomes of multiple model organisms [28] as well as primary human cells, including pluripotent

On or off-target?

The idea to precisely modify complex genomes with designer nucleases can be hampered by insufficient specificity of their activity [47]. The cleavage of nucleases at undesired locations is commonly referred to as off-target activity. Two recent studies have described genome-wide approaches to assess the activity of ZFNs in human cells [48•, 49•] and demonstrated that ZFNs reveal off-target cleavage at several genomic loci that share a certain degree of homology with the canonical target site.

Conclusions

The ability to modify complex genomes with designer nucleases ad libitum has had a significant impact on basic research, biotechnology and human gene therapy. It has opened unprecedented ways to dissect gene function in model organisms, allowed for the production of isogenic cell lines that serve as a source to model genetic disease, and opened novel avenues for personalized therapy of inherited or acquired disorders. Designer nucleases have recently reached the clinic with the generation of

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Work in the laboratory is supported by the German Research Foundation (SPP1230–Ca311/2 and SFB738–C9), the Federal Ministry of Education and Research (InTherGD–01GU0834 and ReGene–01GN1003B), the European Commission's 7th Framework Programme (PERSIST–222878 and HeMiBio–266777), and the Mukoviszidose Institut gGmbH (S03/11).

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