title: Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells.
abstract: Engineered genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. We present a robust, general, scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE), to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on recombinases under the control of a single promoter, which integrates circuit signals on a single transcriptional layer. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells and devised a quantitative, vector-proximity metric to evaluate their performance. Of 113 circuits analyzed, 109 functioned (96.5%) as intended without optimization. The circuits, which are available through Addgene, include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; circuits with small-molecule-inducible control; and circuits that incorporate CRISPR-Cas9 to regulate endogenous genes. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
authors.forename: Benjamin H
authors.initials: BH
authors.lastname: Weinberg
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
authors.forename: N T Hang
authors.initials: NTH
authors.lastname: Pham
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
authors.forename: Leidy D
authors.initials: LD
authors.lastname: Caraballo
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lozanoski
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.|Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
authors.forename: Adrien
authors.initials: A
authors.lastname: Engel
authors.affiliation: Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts, USA.
authors.forename: Swapnil
authors.identifier: 0000-0003-1079-7570
authors.initials: S
authors.lastname: Bhatia
authors.affiliation: Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, Massachusetts, USA.
authors.forename: Wilson W
authors.initials: WW
authors.lastname: Wong
mesh_terms: D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D003198:Computer Simulation; D039301:Computers, Molecular; D053263:Gene Regulatory Networks / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008957:Models, Genetic; D020543:Proteome / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D020543:Proteome