Integrated Circuit Fabrication for Mammalian Cell Biocomputers

Synthetic biological circuits have demonstrated incredible promise for engineering complex, logical control over gene expression. Recently, the Boolean Logic and Arithmetic through DNA Excision (BLADE) single-layer circuit design has enabled the fabrication of highly complex circuits in mammalian cells using site-specific recombinases (SSRs). While SSR-based logic circuits are in valuable for cell-based computation, their digital (on or off) behavior lacks the ability to finely tune gene expression levels. Alternatively, programmable transcription factors offer user-defined gene expression levels but their implementation into circuits has thus far been limited to 2-input logic gates. To bridge the gap between programmability and logic processing, we present the Computation via Recombinase Assisted Transcriptional Effectors (CREATE) platform for multi-input-multi-output (MIMO) integrated circuit design. CREATE uses transcription factors as molecular wiring between layers of BLADE circuits, and has been optimized for use with both dCas9 and zinc finger transcriptional activators. We have established design rules and an extensive promoter library for the facile construction of multiple CREATE circuits in mammalian cells. To demonstrate the power of this design, we present two of the most complex mammalian gene circuits to date: several 2-input digital-to-analog converters (DACs) and the first 4-input 12-output genetic multiplier circuit. CREATE provides anew suite of tools for improved MIMO biocomputation, and will be useful for multiple research applications including the interrogation of complex diseases and drug screening involving multi-pathway regulation.