Overcome the impact of methylated DNA on TALEN™ activity

TALEN™ CpG is the unique service form Cellectis bioresearch to overcome the impact of methylated cytosine on TALEN™ activity.

Validation of your choice

TALEN™ CpG’s activity can be validated at the level of your choice (no validation, on episomal or chromosomal target sequence). We offer standard validation levels for human, mouse & rat cell lines, as well upon request validate the TALEN™ activity on your cell line. Talk to us about your TALEN™ project and let us define the best TALEN™ for your project.

See below the different validation levels:

*immortalized cell lines
**additional 4 weeks for validation report

Find below a list of our TALEN™ CpG options:

Specific solutions for gene modification and knock-out

Do you have other gene editing projects in mind? Learn more on TALEN™ Basic for any gene modification or on TALEN™ Sure KO to ensure knock-out with the 1st coding exon of your gene of interest.

Custom TALEN™ Project Support

For any of your gene editing projects, we offer Custom TALEN™ Project Support, a comprehensive service for TALEN™ project optimization. Comes with dedicated project support managers who will share with you competence and know-how to define the best Custom TALEN™ suited to your genome customization project.

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About methylation

DNA methylation is indispensable for vertebrate genome function and involved in diverse genomic processes such as gene regulation, chromosomal stability, and parental imprinting. DNA methylation is an epigenetic, heritable, stable post-synthesis DNA modification, resulting in the addition of a methyl group at the carbon 5- position of the cytosine ring. The interest in the function of DNA methylation is further heightened by the various human diseases associated with epigenetic dysfunction, a notable example being cancer (1).

It has been recently reported that engineered TALE DNA binding domains are affected by the presence of a 5-methylated cytosine in their endogenous target (2). Even though most cytosine methylation occurs in the context of CpG dinucleotides (70% of CpG dinucleotides are methylated in mammalian and plant somatic/pluripotent cells (3, 4)), it has also been reported in non CpG dinucleotides (5). Moreover, the 5-methylated cytosine has been identified in CpG islands embedded in many promoters (6) and, to a higher extent, in proximal exons of several genes (7). These two critical regulatory regions are generally chosen by investigators to knock-out genes of therapeutic and biotechnological interest using TALEN™ technologies.

How does TALEN™ CpG overcome methylation?

We described recently that TALE DNA binding domains bearing the TALE repeats HD are likely to be sensitive to cytosine methylation, and we could show that substituting HD repeats by N* repeats help to overcome methylation sensitivity (8).

TALEN™ CpG includes the design of a Custom TALEN™ by taking advantage of the TALE repeat N*, which makes TALEN™ CpG DNA insensitive to cytosine methylation. HD/N* substitution within TALE DNA binding domains does not increase TALEN™-induced toxicity, as we demonstrated in CHO cells.

Gene editing becomes more accessible to therapeutic applications, cancer studies and stem cell research

> Discuss with our dedicated Customer Support team your TALEN™ design options.

References:

1. Rakyan, V.K. et al. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project.PLoS Biol 2, e405 (2004).

2. Bultmann, S. et al. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res 40, 5368-5377.

3. Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33 Suppl, 245-254 (2003).

4. Vanyushin, B.F. & Ashapkin, V.V. DNA methylation in higher plants: past, present and future. Biochim Biophys Acta 1809, 360-368.

5. Ramsahoye, B.H. et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci U S A 97, 5237-5242 (2000).

6. Maunakea, A.K. et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253-257.

7. Brenet, F. et al. DNA methylation of the first exon is tightly linked to transcriptional silencing. PLoS One 6, e14524.

8. Valton, J. et al. Overcoming TALE DNA Binding Domain Sensitivity to Cytosine Methylation. J Biol Chem.