STAR: a simple TAL effector assembly reaction using isothermal assembly.
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.
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK.
Sabine
S
Gogolok
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK.
Claudia
C
Garcia-Diaz
MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK.
Steven M
SM
Pollard