TAL Effector Modulators: Beyond Nucleases

Programmable DNA-binding proteins such as Zinc Finger Nucleases, TALEs and CRISPR/Cas9 are versatile tools for genome modification. While mutagenesis (knock outs and knock ins) has been a major initial objective in using these systems to date, it is clear that they can also be used in other ways.

TAL Effector Domains can be switched to achieve a variety of functions and activities.

TAL Effector Domains can be switched to achieve a variety of functions and activities.

Zhang et al (2014) describe their efforts to understand some of the rules for using TALEs as transcriptional activators or repressors (they are not the first). TALEs, because of their programmable DNA binding properties, can be linked to other functional protein domains besides nucleases to create DNA binding proteins with other functionalities. For example, if TALEs are fused with VP16/64 and programmed to bind upstream of a gene it can recruit transcription initiation complexes to initiate transcription.

It is worth noting that CRISPRs can also be used this way but a guide RNA is required for specificity.  If one is considering creating a transgenic organism with modulated gene expression, TALEs are a good choice since it requires only the expression of a programmed protein.

A TALE linked to a transcription activation domain (VP64) to function as a transcription factor.

A TALE linked to a transcription activation domain (VP64) to function as a transcription factor.

 

 

Zhang et al (2014) show that the positioning of the TALE-VPs relative to the transcriptional start makes a big difference in determining the strength of transcription. It was not a simple relationship like close is better than far. They also found that chromosome structure plays a determining role.

Overall they defined 2 rules.
1) The accessibility of the target sequence strongly determines the strength of activation.
2) Target sites should be located within 1.5 kilobases of the transcription start site to effectively activate gene expression.

Zhang et al (2014) also developed a novel type of TALE repressor. There are currently a number of approaches to silencing genes in vivo – antisense RNA and RNAi to name two. TALEs have also been used by adding a “repressor domain” instead of the VP16 activation domain or a nuclease. Zhang et al (2014) have done something a bit different. They have used two TALEs designed to have strong affinity for each other and programmed to bind to neighboring DNA regions. Targeting this pair of TALEs to where either transcription factors or RNA polymerase II interact with DNA can occlude the DNA and prevent transcription. The levels of silencing the report was comparable to siRNA silencing.

TALE transcription activators can be useful in performing mis-expression studies which can assist in determining the function of genes.

TALE-based modulators in combination with transgenic technologies enable the creation of genotypes in ways that may not be otherwise possible for the system of interest.

Insect biologists thinking about what kinds of genetic modifications can be made should be aware of this powerful technology.

 

Zhang H, Li J, Hou S, Wang G, Jiang M, Sun C, Hu X, Zhuang F, Dai  Z, Dai J, Xi JJ (2014) Engineered TAL Effector modulators for the large-scale gain-of-function screening. Nucleic Acids Res doi 10.1093/nar/gku535

 

Recent Related Posts in Technology Topics:

Synthetic Selfish DNA as Genetic Technologies

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