An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device’s output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is competition among genes for shared cellular resources, such as those required for transcription and translation, which can induce ‘coupling’ among otherwise independently-regulated genes. Here, we quantify the effects of resource sharing on engineered genetic systems in mammalian cells and develop an endoribonuclease-based incoherent feedforward loop (iFFL) to make gene expression levels robust to changes in resource availability. Our iFFL accurately controls gene expression levels in various cell lines and in the presence of significant resource sequestration by transcriptional activators. In addition to mitigating resource sharing, our iFFL also adapts gene expression to multiple log decades of DNA copy number variation, substantially improving upon previously-described miRNA-based iFFLs. Ultimately, our iFFL device will enable predictable, robust, and context-independent control of gene expression in mammalian cells.

[1]  D J Segal,et al.  Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Theresa A. Storm,et al.  Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways , 2006, Nature.

[3]  Yohei Yokobayashi,et al.  Aptamer-based and aptazyme-based riboswitches in mammalian cells , 2019, Current Opinion in Chemical Biology.

[4]  Craig M. Crews,et al.  Induced protein degradation: an emerging drug discovery paradigm , 2016, Nature Reviews Drug Discovery.

[5]  Stan J. J. Brouns,et al.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes , 2008, Science.

[6]  Alexander P. S. Darlington,et al.  Engineering Translational Resource Allocation Controllers: Mechanistic Models, Design Guidelines, and Potential Biological Implementations , 2018, bioRxiv.

[7]  Kwang-Hyun Cho,et al.  The biphasic behavior of incoherent feed-forward loops in biomolecular regulatory networks. , 2008, BioEssays : news and reviews in molecular, cellular and developmental biology.

[8]  Yinjie J. Tang,et al.  Decoupling Resource-Coupled Gene Expression in Living Cells. , 2017, ACS synthetic biology.

[9]  Domitilla Del Vecchio,et al.  Modular Analysis and Design of Biological Circuits. , 2019, Current opinion in biotechnology.

[10]  Domitilla Del Vecchio,et al.  Modularity, context-dependence, and insulation in engineered biological circuits. , 2015, Trends in biotechnology.

[11]  P Chambon,et al.  Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1 , 1993, Molecular and cellular biology.

[12]  Rajamanickam Murugan,et al.  Theory on the Dynamics of Feedforward Loops in the Transcription Factor Networks , 2012, PloS one.

[13]  Ron Weiss,et al.  Isocost Lines Describe the Cellular Economy of Genetic Circuits , 2015, Biophysical journal.

[14]  J M Hardwick,et al.  The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16 , 1992, Journal of virology.

[15]  D. Fuhrer,et al.  Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder , 2008, Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme.

[16]  Ron Weiss,et al.  A ‘poly-transfection’ method for rapid, one-pot characterization and optimization of genetic systems , 2019, Nucleic acids research.

[17]  Vanja Haberle,et al.  Transcriptional cofactors display specificity for distinct types of core promoters , 2019, Nature.

[18]  V. Rivera,et al.  Transcriptional squelching re-examined , 1997, Nature.

[19]  A Farr,et al.  A pitfall of using a second plasmid to determine transfection efficiency. , 1992, Nucleic acids research.

[20]  Neda Bagheri,et al.  The COMET toolkit for composing customizable genetic programs in mammalian cells , 2019, bioRxiv.

[21]  Ron Weiss,et al.  Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells , 2014, ACS synthetic biology.

[22]  José Utrilla,et al.  Trade-offs between gene expression, growth and phenotypic diversity in microbial populations , 2020, Current opinion in biotechnology.

[23]  Gábor Balázsi,et al.  Evolutionary regain of lost gene circuit function , 2019, Proceedings of the National Academy of Sciences.

[24]  Ilpo Vattulainen,et al.  An efficient auxin-inducible degron system with low basal degradation in human cells , 2019, Nature Methods.

[25]  Domitilla Del Vecchio,et al.  Modularity, context-dependence, and insulation in engineered biological circuits , 2015 .

[26]  Timothy K Lu,et al.  A multi-landing pad DNA integration platform for mammalian cell engineering , 2018, Nucleic acids research.

[27]  Mark Ptashne,et al.  Negative effect of the transcriptional activator GAL4 , 1988, Nature.

[28]  P. Baeuerle,et al.  The p65 subunit is responsible for the strong transcription activating potential of NF‐kappa B. , 1991, The EMBO journal.

[29]  Anne Gatignol,et al.  Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC , 2007, Nucleic acids research.

[30]  S. Berger,et al.  Genetic isolation of ADA2: A potential transcriptional adaptor required for function of certain acidic activation domains , 1992, Cell.

[31]  Vladimir B. Bajic,et al.  TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins , 2010, Nucleic Acids Res..

[32]  Enoch Yeung,et al.  Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks. , 2017, Cell systems.

[33]  A. Arkin,et al.  Contextualizing context for synthetic biology – identifying causes of failure of synthetic biological systems , 2012, Biotechnology journal.

[34]  T. Hope,et al.  Woodchuck Hepatitis Virus Contains a Tripartite Posttranscriptional Regulatory Element , 1998, Journal of Virology.

[35]  Dylan J. Taatjes,et al.  The Mediator complex and transcription regulation , 2013, Critical reviews in biochemistry and molecular biology.

[36]  S. McKnight,et al.  Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. , 1988, Genes & development.

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

[38]  Gábor Balázsi,et al.  Multiplexed Gene Expression Tuning with Orthogonal Synthetic Gene Circuits , 2020, ACS synthetic biology.

[39]  Adam P. Arkin,et al.  Engineering robust control of two-component system phosphotransfer using modular scaffolds , 2012, Proceedings of the National Academy of Sciences.

[40]  Jacob Beal,et al.  A Method for Fast, High-Precision Characterization of Synthetic Biology Devices , 2012 .

[41]  Reinhard Klein,et al.  WPRE-mediated enhancement of gene expression is promoter and cell line specific. , 2006, Gene.

[42]  Elisabeth Scheer,et al.  Distinct classes of transcriptional activating domains function by different mechanisms , 1990, Cell.

[43]  Domitilla Del Vecchio,et al.  A quasi-integral controller for adaptation of genetic modules to variable ribosome demand , 2018, Nature Communications.

[44]  Christopher A. Voigt,et al.  Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates , 2013, Nature chemical biology.

[45]  T. Tuller,et al.  Modelling and measuring intracellular competition for finite resources during gene expression , 2019, Journal of the Royal Society Interface.

[46]  Timothy K Lu,et al.  Gene networks that compensate for crosstalk with crosstalk , 2019, Nature Communications.

[47]  Gerald Stampfel,et al.  Transcriptional regulators form diverse groups with context-dependent regulatory functions , 2015, Nature.

[48]  Christopher A. Voigt,et al.  Engineered promoters enable constant gene expression at any copy number in bacteria , 2018, Nature Biotechnology.

[49]  Zhen Xie,et al.  Molecular Systems Biology Peer Review Process File Synthetic Incoherent Feed-forward Circuits Show Adaptation to the Amount of Their Genetic Template. Transaction Report , 2022 .

[50]  I. Martins,et al.  Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[51]  Declan G. Bates,et al.  Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes , 2017, Nature Communications.

[52]  Georg Seelig,et al.  MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations. , 2014, ACS synthetic biology.

[53]  Roger D. Kornberg,et al.  A mediator required for activation of RNA polymerase II transcription in vitro , 1991, Nature.

[54]  Guy-Bart Stan,et al.  Characterization, modelling and mitigation of gene expression burden in mammalian cells , 2019, bioRxiv.

[55]  J. P. Ferreira,et al.  Tuning gene expression with synthetic upstream open reading frames , 2013, Proceedings of the National Academy of Sciences.

[56]  Ron Weiss,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2014, Nature Methods.

[57]  M. Gossen,et al.  Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. , 1997, Nucleic acids research.

[58]  Vadim Y Arshavsky,et al.  Proteasome overload is a common stress factor in multiple forms of inherited retinal degeneration , 2013, Proceedings of the National Academy of Sciences.

[59]  William F Marzluff,et al.  Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4 , 2015, RNA.

[60]  Domitilla Del Vecchio,et al.  Retroactivity controls the temporal dynamics of gene transcription. , 2013, ACS synthetic biology.

[61]  Ping Wang,et al.  Cellular toxicity induced by SRF-mediated transcriptional squelching. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[62]  Christopher A. Voigt,et al.  Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors , 2018, Nature Chemical Biology.

[63]  Zhen Xie,et al.  Modular construction of mammalian gene circuits using TALE transcriptional repressors , 2014, Nature chemical biology.

[64]  Roger D. Kornberg,et al.  A novel mediator between activator proteins and the RNA polymerase II transcription apparatus , 1990, Cell.

[65]  S. Berger,et al.  Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: Evidence for transcriptional adaptors , 1990, Cell.

[66]  Y. Benenson,et al.  Synthetic control systems for high performance gene expression in mammalian cells , 2018, Nucleic acids research.

[67]  Olivier Elemento,et al.  5′ UTR m6A Promotes Cap-Independent Translation , 2015, Cell.

[68]  M. Moore,et al.  Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides , 2015, Cell.

[69]  M. Manhart,et al.  Stress-response balance drives the evolution of a network module and its host genome , 2015, Molecular systems biology.

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

[71]  Lily Shiue,et al.  Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing. , 2013, Molecular cell.

[72]  Deepak Mishra,et al.  A load driver device for engineering modularity in biological networks , 2014, Nature Biotechnology.

[73]  James J. Collins,et al.  Comparative Analysis of Cas9 Activators Across Multiple Species , 2016, Nature Methods.

[74]  Jun Ma,et al.  GAL4-VP16 is an unusually potent transcriptional activator , 1988, Nature.

[75]  Christopher A. Voigt,et al.  Genetic circuit design automation , 2016, Science.

[76]  Alexander M. Samsonov,et al.  The dynamics of a feed-forward loop depends on the regulator type in its indirect pathway , 2015 .

[77]  Breanna DiAndreth,et al.  PERSIST: A programmable RNA regulation platform using CRISPR endoRNases , 2019, bioRxiv.

[78]  D. C. Baird,et al.  Experimentation: An Introduction to Measurement Theory and Experiment Design , 1965 .

[79]  Ron Weiss,et al.  A platform for rapid prototyping of synthetic gene networks in mammalian cells , 2014, Nucleic acids research.

[80]  Domitilla Del Vecchio,et al.  Resource Competition Shapes the Response of Genetic Circuits. , 2017, ACS synthetic biology.

[81]  Michael Z. Lin,et al.  Tunable and reversible drug control of protein production via a self-excising degron , 2015, Nature chemical biology.

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