Programmable and Highly Resolved In Vitro Detection of 5‐Methylcytosine by TALEs

Abstract

Gene expression is extensively regulated by specific patterns of genomic 5-methylcytosine (mC), but the ability to directly detect this modification at user-defined genomic loci is limited. One reason is the lack of molecules that discriminate between mC and cytosine (C) and at the same time provide inherent, programmable sequence-selectivity. Programmable transcription-activator-like effectors (TALEs) have been observed to exhibit mC-sensitivity in vivo, but to only a limited extent in vitro. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome.

Synthetic epigenetics—towards intelligent control of epigenetic states and cell identity

“…Importantly, the known mammalian DNA modifications, 5-methylcytosine (5mC) and its oxidation products are also presented in the major groove of DNA and therefore can influence the binding of these domains to DNA [91,92]. There are examples of both natural and synthetic zinc finger proteins that recognize 5-methylated cytosine embedded in a specific DNA context [93].…”

“…The modification state sensitivity of the zinc finger and TALE arrays should be taken into consideration while designing experiments, especially when the methylation status of the targeted region is not known. Additionally, certain TALE repeats have been reported to be insensitive to 5mC modification and therefore could be employed to overcome this limitation [91]. …”

Epigenetic editing: How cutting-edge targeted epigenetic modification might provide novel avenues for autoimmune disease therapy

“…Their large size and lack of an endogenous analogue has led many to worry about potential immunogenicity if used in vivo in human patients. A final challenge, at least from an epigenetic editing perspective, is that dense local DNA methylation inhibits the binding of most TALEs, limiting their usefulness in editing techniques aimed at reducing DNA methylation [16]. Nonetheless, TALEs have been successfully used for epigenetic editing in a number of studies.…”

Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will

“…Moreover, their big sizes and immunogenicity likely will hamper their uses in clinical applications. Likewise, DNA methylation has been shown to hamper the binding of TALEs, restricting their accessibility at heterochromatin regions [44]. …”