title: Two- and three-input TALE-based AND logic computation in embryonic stem cells.
abstract: Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
authors: Florian
authors: F
authors: Lienert
authors: Joseph P
authors: JP
authors: Torella
authors: Jan-Hung
authors: JH
authors: Chen
authors: Michael
authors: M
authors: Norsworthy
authors: Ryan R
authors: RR
authors: Richardson
authors: Pamela A
authors: PA
authors: Silver
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism*; D053263:Gene Regulatory Networks; D006801:Humans; D047668:Inteins; D051379:Mice; D019154:Protein Splicing; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D015534:Trans-Activators
references: Proc Natl Acad Sci U S A. 2010 May 4;107(18):8117-22
references: 20404201
references: Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9226-31
references: 9689062
references: Nucleic Acids Res. 2011 Jul;39(12):e82
references: 21493687
references: Science. 2011 Sep 2;333(6047):1307-11
references: 21885784
references: Nat Methods. 2013 Mar;10(3):239-42
references: 23377379
references: Science. 2007 Oct 26;318(5850):645-8
references: 17962564
references: Genes Dev. 2012 Jul 1;26(13):1486-97
references: 22751502
references: Nat Rev Cancer. 2011 Dec 01;12(1):51-8
references: 22129804
references: Annu Rev Phytopathol. 2010;48:419-36
references: 19400638
references: Cell. 2012 Aug 3;150(3):647-58
references: 22863014
references: Nat Genet. 2011 Oct 02;43(11):1091-7
references: 21964573
references: Nucleic Acids Res. 2012 Aug;40(15):7584-95
references: 22581776
references: Nat Methods. 2008 May;5(5):374-5
references: 18446154
references: Curr Opin Chem Biol. 2012 Aug;16(3-4):355-61
references: 22609142
references: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14628-33
references: 9843940
references: Science. 2009 Dec 11;326(5959):1509-12
references: 19933107
references: Science. 2012 Feb 10;335(6069):716-9
references: 22223736
references: Hum Gene Ther. 2012 Aug;23(8):883-90
references: 22587705
references: Science. 2008 Oct 17;322(5900):456-60
references: 18927397
references: Genome Biol. 2012 Feb 20;13(2):240
references: 22348749
references: J Virol. 2011 Sep;85(18):9268-75
references: 21752918
references: Nat Rev Genet. 2012 Jun 12;13(7):455-68
references: 22688678
references: Science. 2009 Dec 11;326(5959):1501
references: 19933106
references: Mol Syst Biol. 2010 Dec 21;6:444
references: 21179016
references: Nat Commun. 2012 Jul 24;3:968
references: 22828628
references: Nature. 2012 Jul 5;487(7405):123-7
references: 22722847
references: Sci Rep. 2012;2:897
references: 23193439
references: Cell. 1993 Aug 13;74(3):505-14
references: 8348617
references: Nat Biotechnol. 2010 Apr;28(4):355-60
references: 20351688
references: J Biol Chem. 2004 Aug 20;279(34):35281-6
references: 15194682
references: Trends Biotechnol. 2013 Mar;31(3):155-68
references: 23245728
references: Appl Microbiol Biotechnol. 2010 Jun;87(2):479-89
references: 20449740
references: Nucleic Acids Res. 2012 Jun;40(11):5180-7
references: 22323524
references: Hum Gene Ther. 2008 Sep;19(9):958-64
references: 18788906