Linear-double-stranded DNA (ldsDNA) based AND logic computation in mammalian cells

Synthetic biology employs engineering principles to redesign biological system for clinical or industrial purposes. The development and application of novel genetic devices for genetic circuits construction will facilitate the rapid development of synthetic biology. Here we demonstrate that mammalian cells could perform two- and three-input linear-double-stranded DNA (ldsDNA) based Boolean AND logic computation. Through hydrodynamic ldsDNA delivery, two-input ldsDNA-base AND-gate computation could be achieved in vivo. Inhibition of DNA-PKcs expression, a key enzyme in non-homologous end joining (NHEJ), could significantly downregulate the intensity of output signals from ldsDNA-based AND-gate. We further reveal that in mammalian cells ldsDNAs could undergo end processing and then perform AND-gate calculation to generate in-frame output proteins. Moreover, we show that ldsDNAs or plasmids with identical overlapping sequences could also serve as inputs of AND-gate computation. Our work establishes novel genetic devices and principles for genetic circuits construction, thus may open a new gate for the development of new disease targeting strategies and new protein genesis methodologies.

[1]  I. Lehman.  DNA ligase: structure, mechanism, and function. , 1974, Science.

[2]  J. Weltman.  The 1987 Nobel Prize for physiology or medicine awarded to molecular immunogeneticist Susumu Tonegawa. , 1988, Allergy proceedings : the official journal of regional and state allergy societies.

[3]  T. Lindahl,et al.  Mammalian DNA ligases. , 1992, Annual review of biochemistry.

[4]  T. Komori,et al.  Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. , 1993, Science.

[5]  B. Nadel,et al.  Nucleotide deletion and P addition in V(D)J recombination: a determinant role of the coding-end sequence , 1997, Molecular and cellular biology.

[6]  P. Baumann,et al.  Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. , 1998, Trends in biochemical sciences.

[7]  Yunmei Ma,et al.  Hairpin Opening and Overhang Processing by an Artemis/DNA-Dependent Protein Kinase Complex in Nonhomologous End Joining and V(D)J Recombination , 2002, Cell.

[8]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[9]  D. Schatz.  V(D)J recombination , 2002, Immunological reviews.

[10]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[11]  E. Andrianantoandro,et al.  Synthetic biology: new engineering rules for an emerging discipline , 2006, Molecular systems biology.

[12]  S. Tonegawa.  Somatic generation of immune diversity , 1988, In Vitro Cellular & Developmental Biology.

[13]  J. Liao,et al.  Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels , 2008, Nature.

[14]  Priscilla E. M. Purnick,et al.  The second wave of synthetic biology: from modules to systems , 2009, Nature Reviews Molecular Cell Biology.

[15]  Thomas H Segall-Shapiro,et al.  Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome , 2010, Science.

[16]  B. González,et al.  Modular system for the construction of zinc-finger libraries and proteins , 2010, Nature Protocols.

[17]  C. Bashor,et al.  Rewiring cells: synthetic biology as a tool to interrogate the organizational principles of living systems. , 2010, Annual review of biophysics.

[18]  M. Elowitz,et al.  Build life to understand it , 2010, Nature.

[19]  G. Church,et al.  Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. , 2011, Nature biotechnology.

[20]  R. Weiss,et al.  Multi-input Rnai-based Logic Circuit for Identification of Specific , 2022 .

[21]  J. Collins,et al.  Synthetic Biology Moving into the Clinic , 2011, Science.

[22]  A. Bogdanove,et al.  TAL Effectors: Customizable Proteins for DNA Targeting , 2011, Science.

[23]  P. Silver,et al.  A tunable zinc finger-based framework for Boolean logic computation in mammalian cells , 2012, Nucleic acids research.

[24]  Markus Wieland,et al.  Engineering molecular circuits using synthetic biology in mammalian cells. , 2012, Annual review of chemical and biomolecular engineering.

[25]  A. Casadevall,et al.  A new synthesis for antibody-mediated immunity , 2011, Nature Immunology.

[26]  Shaoduo Yan,et al.  High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[27]  Ahmad S. Khalil,et al.  A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions , 2012, Cell.

[28]  Yen-Hsiang Wang,et al.  Synthetic biology: advancing the design of diverse genetic systems. , 2013, Annual review of chemical and biomolecular engineering.

[29]  Christopher A. Voigt,et al.  Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression. , 2013, Current opinion in chemical biology.

[30]  Cindy Follonier,et al.  Friedreich's ataxia–associated GAA repeats induce replication-fork reversal and unusual molecular junctions , 2013, Nature Structural &Molecular Biology.

[31]  Timothy K Lu,et al.  Synthetic circuits integrating logic and memory in living cells , 2013, Nature Biotechnology.

[32]  William I. Bacchus,et al.  Biomedically relevant circuit-design strategies in mammalian synthetic biology , 2013, Molecular systems biology.

[33]  Ryan R. Richardson,et al.  Two- and three-input TALE-based AND logic computation in embryonic stem cells , 2013, Nucleic acids research.

[34]  F. Lienert,et al.  Synthetic biology in mammalian cells: next generation research tools and therapeutics , 2014, Nature Reviews Molecular Cell Biology.

[35]  Z. Cai,et al.  Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells , 2014, Nature Communications.

[36]  Christopher A. Voigt,et al.  Principles of genetic circuit design , 2014, Nature Methods.

[37]  J. Collins,et al.  A brief history of synthetic biology , 2014, Nature Reviews Microbiology.

[38]  David J. Chen,et al.  DNA-PK: a dynamic enzyme in a versatile DSB repair pathway. , 2014, DNA repair.

[39]  J. Collins,et al.  Synthetic biology devices for in vitro and in vivo diagnostics , 2015, Proceedings of the National Academy of Sciences.

[40]  Alexandro E. Trevino,et al.  Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex , 2014, Nature.

[41]  Mathieu Morel,et al.  Cellular heterogeneity mediates inherent sensitivity–specificity tradeoff in cancer targeting by synthetic circuits , 2016, Proceedings of the National Academy of Sciences.

[42]  D. G. Gibson,et al.  Design and synthesis of a minimal bacterial genome , 2016, Science.

[43]  Christopher A. Voigt,et al.  Synthetic biology to access and expand nature's chemical diversity , 2016, Nature Reviews Microbiology.

[44]  Dacheng Ma,et al.  Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells , 2016, Nature Communications.

[45]  Kole T. Roybal,et al.  Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits , 2016, Cell.

[46]  Claudia C. Wehrspaun,et al.  Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy , 2017, Cell.

[47]  Swapnil Bhatia,et al.  A single-layer platform for Boolean logic and arithmetic through DNA excision in mammalian cells , 2017, Nature Biotechnology.

[48]  Ronja Biehs,et al.  DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination , 2017, Molecular cell.