Cell-Free Multistep Gene Regulatory Cascades Using Eukaryotic ON-Riboswitches Responsive to Expressed Protein Ligands.

One of the most pressing challenges in cell-free synthetic biology is to assemble well-controlled genetic circuits. However, no complex circuits have been reported in eukaryotic cell-free systems, unlike the case in bacterial ones, despite several unique advantages of the former. We here developed protein-responsive upregulating riboswitches (ON-riboswitches) that function in wheat germ extract to create multistep gene regulatory cascades.