Abstract
Transcription activator-like effectors (TALEs) contain modular programmable DNA binding domains. Fusing TALEs with effector domains creates synthetic transcription factors (TALE-TFs) or nucleases (TALENs), enabling precise gene manipulations. The construction of TALEs remains challenging due to their repetitive sequences. Here we report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly (‘Gibson assembly’) that is labour- and cost-effective, accessible, rapid and scalable. A small 68-part fragment library is employed, and the specific TALE repeat sequence is generated within ~8 hours. Sequence-verified TALENs or TALE-TF plasmids targeting 17 bp target sequences can be produced within three days, without the need for stepwise intermediate plasmid production. We demonstrate the utility of STAR through production of functional TALE-TFs capable of activating human SOX2 expression. STAR addresses some of the shortcomings of existing Golden Gate or solid-phase assembly protocols and enables routine production of TALE-TFs that will complement emerging CRISPR/Cas9-based reagents across diverse applications in mammalian stem cell and synthetic biology.
Highlights
We considered whether GA could be exploited to assemble Transcription activator-like effectors (TALEs) DNA binding domains
We report a new approach to TAL effector assembly, which we term simple TALE assembly reaction (STAR)
We have focussed on mammalian cells, but STAR could readily be modified for other organisms or cell lines
Summary
STAR addresses some of the shortcomings of existing Golden Gate or solid-phase assembly protocols and enables routine production of TALE-TFs that will complement emerging CRISPR/Cas9based reagents across diverse applications in mammalian stem cell and synthetic biology. The recent emergence of programmable DNA binding proteins, built upon transcription activator-like effector proteins (TALEs) and CRISPR/Cas[9] architectures, has heralded a new era of simple and efficient genome and chromatin editing. These tools provide the foundations for new approaches to programming gene function and gene regulatory circuits across a multitude of model organisms and cell lines[1]. STAR will help drive forward TALE-based applications to complement CRISPR/Cas reagents
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