Comprehensive analysis of the specificity of transcription activator-like effector nucleases

Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien; Thomas, Séverine; Stella, Stefano; Maréchal, Alan; Langevin, Stéphanie; Benomari, Nassima; Bertonati, Claudia; Silva, George H.; Daboussi, Fayza; Epinat, Jean‐Charles; Montoya, Guillermo; Duclert, Aymeric; Duchâteau, Philippe

doi:10.1093/nar/gku155

Cited by 89 publications

(89 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although each finger module is highly specific to its corresponding trinucleotide sequence, ZFN suffers from context-dependent binding preference when individual modules are combined in an array (66). TALENs also exhibit context-dependent specificity, to a lesser extent (55). In addition, the assembly of TALEN genes encoding large numbers of TALE modules presents a challenge for molecular cloning as well as for efficient viral delivery to target cells (44).…”

Section: Targeted Genome Editing In Mammalian Cellsmentioning

confidence: 99%

CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems

Jiang

Marraffini

2015

Annu. Rev. Microbiol.

175

152

View full text Add to dashboard Cite

Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields.

show abstract

Section: Targeted Genome Editing In Mammalian Cellsmentioning

confidence: 99%

CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems

Jiang

Marraffini

2015

Annu. Rev. Microbiol.

175

152

View full text Add to dashboard Cite

show abstract

“…Compared to ZFNs, TALENs possess a broader targeting range and are less difficult to engineer Doyle et al, 2012). 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).…”

mentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

show abstract

“…In this respect, TALEs differ from other DNA-binding motifs that exhibit a greater functional complexity and that in turn present richer opportunities for fine-tuning recognition. As created using the natural code, TALENs can specify unintended bases in their binding sites 12,14,20,21 and also cleave nontargeted cellular sequences 14,15,20,22,23 . Although levels of off-target activity have not approached those described for the CRISPR-Cas9 platform 21,[24][25][26] , strategies for eliminating such behavior will likely be required to achieve the full potential of this motif, especially for highly sensitive applications such as human therapeutics.…”

mentioning

confidence: 99%

Improved specificity of TALE-based genome editing using an expanded RVD repertoire

et al. 2015

View full text Add to dashboard Cite

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

show abstract

Comprehensive analysis of the specificity of transcription activator-like effector nucleases

Cited by 89 publications

References 41 publications

CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems

CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems

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

Improved specificity of TALE-based genome editing using an expanded RVD repertoire

Contact Info

Product

Resources

About