The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA

Flade, Sarah; Jasper, Julia; Gieß, Mario; Juhasz, Matyas; Dankers, Andreas; Kubik, Grzegorz; Koch, Oliver; Weinhold, Elmar G.; Summerer, Daniel

doi:10.1021/acschembio.7b00324

Cited by 13 publications

(14 citation statements)

References 53 publications

(102 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In addition, studies on the sensitivity of TALE array to epigenetic modifications (e.g., 5-methylcytosine) highlighted how steric hindrance between the amino acids at positions 12 and 13 and the targeted nucleotide is affecting the recognition and binding of an RVD array to its target sequence. 36 , 37 The overall better understanding of the interaction between the RVD array and the targeted sequence allowed the implementation of such non-conventional RVDs, improving the discrimination between different nucleotides and therefore further increasing the specificity of TALE-based molecular tools. 18 , 19 , 20 …”

Section: Discussionmentioning

confidence: 99%

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

Gautron¹,

Juillerat

Guyot³

et al. 2017

Molecular Therapy - Nucleic Acids

View full text Add to dashboard Cite

Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of “off-the-shelf” CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells’ functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.

show abstract

Section: Discussionmentioning

confidence: 99%

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

Gautron¹,

Juillerat

Guyot³

et al. 2017

Molecular Therapy - Nucleic Acids

View full text Add to dashboard Cite

show abstract

“…GFP tag and C-terminal His 6 tag. TALE proteins were expressed and purified as described previously [43]. Briefly, expressions were performed with single clones of Escherichia coli BL21(DE3) Gold transformed with a TALE expression plasmid in Luria-Bertani (LB) medium supplemented with 50 mg ml 21…”

Section: Materials and Methods (A) Construction Expression And Purifimentioning

confidence: 99%

Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors

Rathi¹,

Maurer²,

Summerer³

2018

Phil. Trans. R. Soc. B

Self Cite

View full text Add to dashboard Cite

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

show abstract

“…This occurred with strand‐selectivity and higher sensitivity than an antibody employed for the same targets ,. Natural and mutant TALE repeats were also analyzed for binding to the pro‐ and eukaryotic epigenetic nucleobase N6‐methyladenine (6 mA) and found to be highly promiscuous …”

Section: Design Of Protein Scaffolds With Selectivity For Oxidized MCmentioning

confidence: 99%

Recognition of Oxidized 5‐Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds

Muñoz‐López

Summerer

2017

The Chemical Record

Self Cite

View full text Add to dashboard Cite

Methylation of genomic cytosine to 5-methylcytosine is a central regulatory element of mammalian gene expression with important roles in development and disease. 5-methylcytosine can be actively reversed to cytosine via oxidation to 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine by ten-eleven-translocation dioxygenases and subsequent base excision repair or replication-dependent dilution. Moreover, the oxidized 5-methylcytosine derivatives are potential epigenetic marks with unique biological roles. Key to a better understanding of these roles are insights into the interactions of the nucleobases with DNA-binding protein scaffolds: Natural scaffolds involved in transcription, 5-methylcytosine-reading and -editing as well as general chromatin organization can be selectively recruited or repulsed by oxidized 5-methylcytosines, forming the basis of their biological functions. Moreover, designer protein scaffolds engineered for the selective recognition of oxidized 5-methylcytosines are valuable tools to analyze their genomic levels and distribution. Here, we review recent structural and functional insights into the molecular recognition of oxidized 5-methylcytosine derivatives in DNA by selected protein scaffolds.

show abstract

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA

Cited by 13 publications

References 53 publications

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors

Recognition of Oxidized 5‐Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds

Contact Info

Product

Resources

About