ABSTRACT
Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.
Subject(s)
Protein Engineering/methods , Trans-Activators/genetics , DNA Breaks, Double-Stranded , DNA Cleavage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyribonucleases, Type II Site-Specific , Endonucleases/genetics , Endonucleases/metabolism , Homeodomain Proteins/genetics , Plant Leaves/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Reproducibility of Results , Nicotiana/geneticsABSTRACT
We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes. The purified transferase was free from poly (ADP-ribose) polymerase activity. The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent. Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa. The optimum pH of the reaction is about 8.0. The K(m) value for NAD+ of the purified enzyme is about 130 microM. Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity. A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified. Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone. We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts. This gene has been expressed in E. coli and ADP-ribosylation activity has been demonstrated using histones as substrate. The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate. Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity. The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes. There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals. We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.
