Deciphering and Exploitation of the Adjacent Transcriptional Regulators for Synthetic Biology

Designable DNA binding domains are an extremely useful tool for the transcriptional regulation of the selected endogenous genes, as well as for the construction of synthetic genetic circuits. Natural transcriptional regulators sometimes act synergistically and can affect binding of other transcriptional regulators; however this phenomenon usually depends on each combination. Here we report that we can affect binding of transcription activator-like effectors (TALEs) by other TALEs with an overlapping or an adjacent recognition site. This occurs in a polarized manner, by preferential binding of the 5â?? TALE, which could be explained by the stronger binding of the RVDs at the TALE N-terminal domain of TALEs. This phenomenon allows introduction of negative regulation by displacing 3` bound TALE-based activators and construction of logic functions with kinetics superior to the traditionally used KRAB domains. We demonstrate the function of this mechanism on up to 5 consecutive TALE target sites and construct all 16 two-input logic gates and a half-adder circuit. Displacement by the adjacent TALEs can also be used to displace other DNA binding domains, such as Cas9-sgRNA.