Two- and three-input AND logic computation using a split TALE

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Florian Lienert, Michael Norsworthy and Pam Silver

Harvard Medical School, United States

The de novo design of Transcriptional Activator-Like Effectors (TALEs) targeting a given sequence is simple and enables the generation of a set of transcription factors that are orthogonal to each other and to a host genome. Thus, TALEs represent potentially attractive components of synthetic gene regulatory circuits. Towards this goal, we here tested whether TALEs can be used for Boolean logic computation in mammalian cells. In order to minimize interference with transcriptional regulation in the host cell, we computationally designed a TALE recognizing a 20 base pair sequence that is orthogonal to all promoter regions in the human and mouse genomes. We then identified an amino acid position within this TALE that is optimal for split intein-mediated protein splicing. Employing this TALE protein splicing strategy we generated two- and three-input AND logic circuits, which show 200 – 600 fold induction of transcriptional activity in transient transfection assays. Using a novel approach for modular assembly of large DNA constructs we integrated and tested variants of these logic circuits at a defined genomic site in mouse embryonic stem cells. Together, these results demonstrate the potential of split TALEs as a novel tool for building transcriptional networks in mammalian cells.