A Multiplexed Transcription Activator-like Effector System for Detecting Specific DNA Sequences

Abstract

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

Targeted DNA Demethylation: Vectors, Effectors and Perspectives

“…The DNA-binding domain of each TALE consists of 34-amino acid repeat modules arranged in tandem that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing [173]. The molecular tool through which DNA demethylase is targeted to the disease-related gene promoters can be made by tethering human TET1 demethylase enzyme to a TALE repeat that has targeted DNAbinding specificities [142].…”