Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation

Abstract

Background: TALE-based technologies are poised to revolutionize the field of biotechnology; however, their sensitivity to cytosine methylation may drastically restrict their ranges of applications. Results: TALE repeat N* proficiently accommodates 5-methylated cytosine. Conclusion: Sensitivity of TALE to cytosine methylation can be overcome by using TALE repeat N*. Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.Transcription activator-like effectors (TALEs), 4 a group of bacterial plant pathogen proteins, have recently emerged as new engineerable scaffolds for production of tailored DNA binding domains with chosen specificities (1). Interest in these systems comes from the apparent simple cipher governing DNA recognition by their DNA binding domain (2, 3). The TALE DNA binding domain is composed of multiple TALE repeats that individually recognize one DNA base pair through specific amino acid di-residues (repeat variable di-residues or RVDs). The remarkably high specificity of TALE repeats and the apparent absence of context-dependent effects among repeats in an array allow modular assembly of TALE DNA binding domains able to recognize almost any DNA sequence of interest. Within the past 2 years, engineered TALE DNA binding domains have been fused to transcription activator (dTALEs) (4), repressor (5), or nuclease domains (TALENs) (6) and used to specifically regulate or modify genes of interest (1). Although successfully used in different cellular contexts, engineered TALE DNA binding domains have recently been reported to be affected by the presence of 5-methylated cytosine (5mC) in their endogenous cognate target (7). Often considered as the fifth base, 5mC is found in about 70% of CpG dinucleotides in mammalian and plant somatic/pluripotent cells (8, 9) and has also been reported in 5-cytosine-phosphoadenine, 5-cytosine-phosphothymine, and 5-cytosine-phosphocytosine dinucleotides (10). Moreover, 5mC has been identified in CpG islands embedded in many promoters (11) and, to a higher extent, in proximal exons of several genes (12). These two critical regulatory regions are generally chosen by investigators to knock out genes of therapeutic and biotechnological interest or to modulate their expression using TALE-based technologies. The ubiquity of 5mC in different cell types and genomic kingdoms, its particular localization, and its negative impact on dTALE activity reported in Ref. 7 make this epigenetic modification a major dra...

The New State of the Art: Cas9 for Gene Activation and Repression

“…However, the use of TALEs in particular can be advantageous when a particular target area lacks a PAM, since TALEs can be programmed to target any sequence. TALEs are able to distinguish between methylcytosine (mC) and cytosine (C), which may be advantageous or disadvantageous, depending on the situation (52,53,63). The methylation status of the targeted site must be taken into account when designing TALEs, which is not the case for Cas9 targeting.…”

A Cut above the Rest: Targeted Genome Editing Technologies in Human Pluripotent Stem Cells

“…The reported sensitivity of TALENs to 5-methylcytosine could be a more serious drawback of the TALEN technology because of the prevalence of this DNA modification in the genome (38,39). Recent evidence shows that this problem may be overcome by engineering 5-methylcytosineinsensitive TALE DNA-binding domains (39). The extent of off-target effect of TALENs is largely unknown.…”

“…Although this should not prohibit successful design of TALENs in most cases, it may be an issue when modifying a specific mutation especially for future cell-based gene therapy. The reported sensitivity of TALENs to 5-methylcytosine could be a more serious drawback of the TALEN technology because of the prevalence of this DNA modification in the genome (38,39). Recent evidence shows that this problem may be overcome by engineering 5-methylcytosineinsensitive TALE DNA-binding domains (39).…”

Mouse Genome Engineering Using Designer Nucleases

“…TALENs have been shown to have a high success rate in targeting a wide range of genes in a number of organisms; however, recent studies suggest that TALEN binding is sensitive to cytosine methylation 30,31 .Thus, newly generated nuclease pairs, for example TALENs cloned into pCAG-T7 vectors, can be transfected transiently into a mouse cell line such as NIH-3T3 or Neuro-2a, which mimic the chromatin state of the mouse embryo to some extent. Here, nuclease activity can be estimated using the T7 endonuclease assay or a restriction digest of PCR product as described in section 5 prior to mRNA synthesis and microinjection.…”