Deciphering Combinatorial Genetics.

High-order interactions among components of interconnected genetic networks regulate complex functions in biological systems, but deciphering these interactions is challenging. New strategies are emerging to decode these combinatorial genetic interactions across a wide range of organisms. Here, we review advances in multiplexed and combinatorial genetic perturbation technologies and high-throughput profiling platforms that are enabling the systematic dissection of complex genetic networks. These rapidly evolving technologies are being harnessed to probe combinatorial gene functions in functional genomics studies and have the potential to advance our understanding of how genetic networks regulate sophisticated biological phenotypes, to generate novel therapeutic strategies, and to enable the engineering of complex artificial gene networks.

[1]  J. Keith Joung,et al.  Robust, synergistic regulation of human gene expression using TALE activators , 2013, Nature Methods.

[2]  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.

[3]  Yoshihide Hayashizaki,et al.  A predictive computational framework for direct reprogramming between human cell types , 2016, Nature Genetics.

[4]  E. Fuchs,et al.  RNAi screens in mice identify physiological regulators of oncogenic growth , 2013, Nature.

[5]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[6]  Y. Dong,et al.  Systematic functional analysis of the Caenorhabditis elegans genome using RNAi , 2003, Nature.

[7]  Roland Arnold,et al.  A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities , 2013, Molecular systems biology.

[8]  Michael B. Elowitz,et al.  Combinatorial gene regulation by modulation of relative pulse timing , 2015, Nature.

[9]  R. Lockey,et al.  Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. , 2015, BioTechniques.

[10]  Matthew C. Canver,et al.  BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis , 2015, Nature.

[11]  J. Harrow,et al.  A conditional knockout resource for the genome-wide study of mouse gene function , 2011, Nature.

[12]  I. Lemischka,et al.  Genomewide gain-of-function genetic screen identifies functionally active genes in mouse embryonic stem cells. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[13]  C. Barbas,et al.  ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. , 2013, Trends in biotechnology.

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

[15]  Ryan T Gill,et al.  Strategy for directing combinatorial genome engineering in Escherichia coli , 2012, Proceedings of the National Academy of Sciences.

[16]  Morgan L. Maeder,et al.  CRISPR RNA-guided activation of endogenous human genes , 2013, Nature Methods.

[17]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[18]  Sourav Bandyopadhyay,et al.  Quantitative genetic-interaction mapping in mammalian cells , 2013, Nature Methods.

[19]  Michael T. McManus,et al.  A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility , 2013, Cell.

[20]  N. Perrimon,et al.  High-throughput RNAi screening in cultured cells: a user's guide , 2006, Nature Reviews Genetics.

[21]  M. Golding,et al.  A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells , 2011, Gene Therapy.

[22]  L. MacNeil,et al.  Gene regulatory networks and the role of robustness and stochasticity in the control of gene expression. , 2011, Genome research.

[23]  J. Joung,et al.  Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition , 2015, Nature Biotechnology.

[24]  Vania Broccoli,et al.  Setting a highway for converting skin into neurons. , 2011, Journal of molecular cell biology.

[25]  Sean C. Bendall,et al.  Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum , 2011, Science.

[26]  Zengrong Zhu,et al.  An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. , 2014, Cell stem cell.

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

[28]  H. Cordell Detecting gene–gene interactions that underlie human diseases , 2009, Nature Reviews Genetics.

[29]  Aviv Regev,et al.  Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing , 2014, Nature Biotechnology.

[30]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[31]  Wolfgang Huber,et al.  Mapping genetic interactions in human cancer cells with RNAi and multiparametric phenotyping , 2013, Nature Methods.

[32]  Sheng Ding,et al.  Genome‐wide gain‐of‐function screen identifies novel regulators of pluripotency , 2010, Stem cells.

[33]  G. Church,et al.  Cas9 gRNA engineering for genome editing, activation and repression , 2015, Nature Methods.

[34]  Yi Jing,et al.  Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs , 2015, Proceedings of the National Academy of Sciences.

[35]  I. Sancho-Martinez,et al.  Lineage conversion methodologies meet the reprogramming toolbox , 2012, Nature Cell Biology.

[36]  R. Maehr,et al.  Functional annotation of native enhancers with a Cas9 -histone demethylase fusion , 2015, Nature Methods.

[37]  R. Weiss,et al.  Foundations for the design and implementation of synthetic genetic circuits , 2012, Nature Reviews Genetics.

[38]  Huiming Ding,et al.  eSGA: E. coli synthetic genetic array analysis , 2008, Nature Methods.

[39]  Anne E Carpenter,et al.  Systematic genome-wide screens of gene function , 2004, Nature Reviews Genetics.

[40]  S. Elledge,et al.  Functional identification of optimized RNAi triggers using a massively parallel sensor assay. , 2011, Molecular cell.

[41]  Simon Hippenmeyer,et al.  DICE, an efficient system for iterative genomic editing in human pluripotent stem cells , 2013, Nucleic acids research.

[42]  L. Busino,et al.  Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis , 2004, Oncogene.

[43]  Christof Fellmann,et al.  A computational algorithm to predict shRNA potency. , 2014, Molecular cell.

[44]  Huiming Ding,et al.  Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics , 2014, Proceedings of the National Academy of Sciences.

[45]  S. Andreadis,et al.  Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector , 2009, Gene Therapy.

[46]  P. Phillips Epistasis — the essential role of gene interactions in the structure and evolution of genetic systems , 2008, Nature Reviews Genetics.

[47]  Michael Boutros,et al.  The art and design of genetic screens: RNA interference , 2008, Nature Reviews Genetics.

[48]  G. Bormans,et al.  Highly efficient multicistronic lentiviral vectors with peptide 2A sequences. , 2009, Human gene therapy.

[49]  C. Bakal,et al.  Genomic screening with RNAi: results and challenges. , 2010, Annual review of biochemistry.

[50]  R. Elkon,et al.  Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9 , 2016, Nature Biotechnology.

[51]  N. Perrimon,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2015, Nature Methods.

[52]  M. Félix,et al.  Pervasive robustness in biological systems , 2015, Nature Reviews Genetics.

[53]  R. Allada,et al.  Emerging roles for post-transcriptional regulation in circadian clocks , 2013, Nature Neuroscience.

[54]  Swapnil Bhatia,et al.  Functional optimization of gene clusters by combinatorial design and assembly , 2014, Nature Biotechnology.

[55]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[56]  S. Yamanaka,et al.  From Genomics to Gene Therapy: Induced Pluripotent Stem Cells Meet Genome Editing. , 2015, Annual review of genetics.

[57]  T. Lu,et al.  Synthetic recombinase-based state machines in living cells , 2016, Science.

[58]  Christopher M. Vockley,et al.  RNA-guided gene activation by CRISPR-Cas9-based transcription factors , 2013, Nature Methods.

[59]  Max A. Horlbeck,et al.  Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation , 2014, Cell.

[60]  A. Burgard,et al.  Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. , 2011, Nature chemical biology.

[61]  Yarden Katz,et al.  Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system , 2013, Cell Research.

[62]  C. Kostic,et al.  Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors , 2015, Molecular therapy. Methods & clinical development.

[63]  Jay Shendure,et al.  Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair , 2014, Nature.

[64]  Gary D Bader,et al.  Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants , 2001, Science.

[65]  J. Rinn,et al.  Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display , 2015, Nature Methods.

[66]  Michael R. Green,et al.  Transcriptional regulatory elements in the human genome. , 2006, Annual review of genomics and human genetics.

[67]  Farren J. Isaacs,et al.  Programming cells by multiplex genome engineering and accelerated evolution , 2009, Nature.

[68]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

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

[70]  T. Lu,et al.  Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas , 2013, ACS synthetic biology.

[71]  Deepak Srivastava,et al.  MicroRNAs as regulators of differentiation and cell fate decisions. , 2010, Cell stem cell.

[72]  Ruhong Zhou,et al.  Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains , 2012, Nature Communications.

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

[74]  Max A. Horlbeck,et al.  Next-generation libraries for robust RNA interference-based genome-wide screens , 2015, Proceedings of the National Academy of Sciences.

[75]  J. Rinn,et al.  lincRNAs act in the circuitry controlling pluripotency and differentiation , 2011, Nature.

[76]  D. Srivastava,et al.  Direct cardiac reprogramming: from developmental biology to cardiac regeneration. , 2013, Circulation research.

[77]  Z. Yakhini,et al.  Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters , 2012, Nature Biotechnology.

[78]  R. Sachidanandam,et al.  High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries , 2012, Nature Methods.

[79]  Y. Buganim,et al.  Nuclear Reprogramming by Defined Factors: Quantity Versus Quality. , 2016, Trends in cell biology.

[80]  M. Rosenfeld,et al.  Brd4 and JMJD6-Associated Anti-Pause Enhancers in Regulation of Transcriptional Pause Release , 2013, Cell.

[81]  S. Hébert,et al.  Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function , 2011, Molecular Neurodegeneration.

[82]  Lihua Julie Zhu,et al.  Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology , 2015, Frontiers in Biology.

[83]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[84]  G. Church,et al.  Identifying regulatory networks by combinatorial analysis of promoter elements , 2001, Nature Genetics.

[85]  J. Glorioso,et al.  A Herpes Simplex Virus Vector System for Expression of Complex Cellular cDNA Libraries , 2010, Journal of Virology.

[86]  A. Pasquinelli MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship , 2012, Nature Reviews Genetics.

[87]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[88]  W. Fairbrother,et al.  Combinatorial binding of transcription factors in the pluripotency control regions of the genome. , 2011, Genome research.

[89]  Y. Ikeda,et al.  Lentiviral vectors: basic to translational. , 2012, The Biochemical journal.

[90]  Rainer Breitling,et al.  Computational tools for the synthetic design of biochemical pathways , 2012, Nature Reviews Microbiology.

[91]  S. Swamy,et al.  Neurotransmitters Drive Combinatorial Multistate Postsynaptic Density Networks , 2009, Science Signaling.

[92]  Thomas Vierbuchen,et al.  Molecular roadblocks for cellular reprogramming. , 2012, Molecular cell.

[93]  Timothy K Lu,et al.  Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM , 2016, Proceedings of the National Academy of Sciences.

[94]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

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

[96]  L. Naldini,et al.  Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters , 2005, Nature Biotechnology.

[97]  D. Bartel MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[98]  K. Gunsalus,et al.  Combinatorial microRNA target predictions , 2005, Nature Genetics.

[99]  Marcello Maresca,et al.  Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining , 2013, Genome research.

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

[101]  David A. Scott,et al.  Rationally engineered Cas9 nucleases with improved specificity , 2015, Science.

[102]  Gary D Bader,et al.  Global Mapping of the Yeast Genetic Interaction Network , 2004, Science.

[103]  S. Carroll,et al.  Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila , 2005, Nature.

[104]  Fyodor D Urnov,et al.  Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases , 2007, Proceedings of the National Academy of Sciences.

[105]  B. Palsson,et al.  Constraining the metabolic genotype–phenotype relationship using a phylogeny of in silico methods , 2012, Nature Reviews Microbiology.

[106]  Ying Zhang,et al.  Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. , 2014, Cell stem cell.

[107]  Feng Zhang,et al.  Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease , 2015, Nature Biotechnology.

[108]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[109]  Neville E. Sanjana,et al.  High-throughput functional genomics using CRISPR–Cas9 , 2015, Nature Reviews Genetics.

[110]  K. Kaestner,et al.  TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts. , 2015, The Journal of clinical investigation.

[111]  Susan R. Wente,et al.  Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system , 2013, Proceedings of the National Academy of Sciences.

[112]  Alex Toftgaard Nielsen,et al.  CRMAGE: CRISPR Optimized MAGE Recombineering , 2016, Scientific Reports.

[113]  Thomas Vierbuchen,et al.  Direct conversion of fibroblasts to functional neurons by defined factors , 2010, Nature.

[114]  Pamela A. Silver,et al.  Engineering synthetic TAL effectors with orthogonal target sites , 2012, Nucleic acids research.

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

[116]  J. Joung,et al.  High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets , 2015, Nature.

[117]  Irving L. Weissman,et al.  Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding , 2011, Nature Biotechnology.

[118]  B. Andrews,et al.  Systematic mapping of genetic interaction networks. , 2009, Annual review of genetics.

[119]  C. Myers,et al.  Genetic interaction networks: toward an understanding of heritability. , 2013, Annual review of genomics and human genetics.

[120]  B. Dickson,et al.  A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila , 2007, Nature.

[121]  J. Bonventre,et al.  The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[122]  N. Grindley,et al.  Mechanisms of site-specific recombination. , 2003, Annual review of biochemistry.

[123]  Lawrence S. Hon,et al.  The roles of binding site arrangement and combinatorial targeting in microRNA repression of gene expression , 2007, Genome Biology.

[124]  R. Kwok Five hard truths for synthetic biology , 2010, Nature.

[125]  Luke A. Gilbert,et al.  Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds , 2015, Cell.

[126]  Juan M. Vaquerizas,et al.  Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. , 2010, Genome research.

[127]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[128]  Paul Shannon,et al.  Derivation of genetic interaction networks from quantitative phenotype data , 2005, Genome Biology.

[129]  T. Lu,et al.  Synthetic biology: an emerging engineering discipline. , 2012, Annual review of biomedical engineering.

[130]  H. Kim,et al.  A guide to genome engineering with programmable nucleases , 2014, Nature Reviews Genetics.

[131]  Juan M. Vaquerizas,et al.  A census of human transcription factors: function, expression and evolution , 2009, Nature Reviews Genetics.

[132]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[133]  Margaret S. Ebert,et al.  MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells , 2007, Nature Methods.

[134]  Toni Cathomen,et al.  Unexpected failure rates for modular assembly of engineered zinc fingers , 2008, Nature Methods.

[135]  Timothy K Lu,et al.  Massively parallel high-order combinatorial genetics in human cells , 2015, Nature Biotechnology.

[136]  Norbert Perrimon,et al.  RNAi screening comes of age: improved techniques and complementary approaches , 2014, Nature Reviews Molecular Cell Biology.

[137]  Kerry Bloom,et al.  Systematic triple-mutant analysis uncovers functional connectivity between pathways involved in chromosome regulation. , 2013, Cell reports.

[138]  Timothy K Lu,et al.  Synthetic analog and digital circuits for cellular computation and memory. , 2014, Current opinion in biotechnology.

[139]  A. Brunet,et al.  The FoxO code , 2008, Oncogene.

[140]  Farren J. Isaacs,et al.  Genomes by design , 2015, Nature Reviews Genetics.

[141]  Samantha A. Morris,et al.  CellNet: Network Biology Applied to Stem Cell Engineering , 2014, Cell.

[142]  E. Furlong,et al.  Transcription factors: from enhancer binding to developmental control , 2012, Nature Reviews Genetics.

[143]  J. Kling Cytometry: Measure for measure , 2015, Nature.

[144]  James C Liao,et al.  Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde , 2009, Nature Biotechnology.

[145]  Eugen C. Buehler,et al.  C911: A Bench-Level Control for Sequence Specific siRNA Off-Target Effects , 2012, PloS one.

[146]  Takanori Kanai,et al.  Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids , 2015, Nature Medicine.

[147]  E. Lander Initial impact of the sequencing of the human genome , 2011, Nature.

[148]  Christopher M. Vockley,et al.  Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers , 2015, Nature Biotechnology.

[149]  Li Li,et al.  MicroRNA-mediated conversion of human fibroblasts to neurons , 2011, Nature.

[150]  T. Hughes,et al.  Mapping pathways and phenotypes by systematic gene overexpression. , 2006, Molecular cell.

[151]  M. Fussenegger,et al.  Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform. , 2007, Biotechnology and bioengineering.

[152]  Ariel S. Schwartz,et al.  An Atlas of Combinatorial Transcriptional Regulation in Mouse and Man , 2010, Cell.

[153]  Dagmar Wirth,et al.  Road to precision: recombinase-based targeting technologies for genome engineering. , 2007, Current opinion in biotechnology.

[154]  Timothy K. Lu,et al.  Continuous genetic recording with self-targeting CRISPR-Cas in human cells , 2016, Science.

[155]  P. Liberali,et al.  Single-cell and multivariate approaches in genetic perturbation screens , 2014, Nature Reviews Genetics.

[156]  Dana Carroll,et al.  Genome engineering with targetable nucleases. , 2014, Annual review of biochemistry.

[157]  Thomas M Green,et al.  A public genome-scale lentiviral expression library of human ORFs , 2011, Nature Methods.

[158]  Julie M. Sahalie,et al.  Supplementary Figure and Table Legends , 2022 .

[159]  H. Federoff,et al.  Extending the transposable payload limit of Sleeping Beauty (SB) using the Herpes Simplex Virus (HSV)/SB amplicon-vector platform , 2010, Gene Therapy.

[160]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[161]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[162]  E. Marder,et al.  Variability, compensation and homeostasis in neuron and network function , 2006, Nature Reviews Neuroscience.

[163]  M. Zavolan,et al.  Identification and consequences of miRNA–target interactions — beyond repression of gene expression , 2014, Nature Reviews Genetics.

[164]  T. Lu,et al.  Genomically encoded analog memory with precise in vivo DNA writing in living cell populations , 2014, Science.

[165]  R. Nussinov,et al.  Allosteric post-translational modification codes. , 2012, Trends in biochemical sciences.