Mapping Genetic Interactions

Description

This volume details methods of identifying synthetic lethal, genetic interactions by various approaches in different model systems including human cancer cells. Chapters guide readers through genetic interactions in model organisms, RNA interference, CRISPR/Cas9 based genome editing technologies, drug-gene interactions, mapping chemical genetic interactions, synergistic drug-gene relations, single cell sequencing, gene expression profiling, and novel genetic interactions. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.   Authoritative and cutting-edge, Genetic Interaction Mapping aims to be a useful practical guide to researches to help further their study in this field. Preface Acknowledgments Contents Contributors About the Editors Part I: Mapping Genetic Interactions in Model Systems Chapter 1: Quantitative Genetic Screens for Mapping Bacterial Pathways and Functional Networks 1 Introduction 2 Materials 2.1 Media, Stock Solutions, and Reagents 2.2 Equipment 2.3 Pinning System, Plates and Accessories for Working with Cultures 2.4 Bacterial Strains and Plasmids 3 Methods 3.1 Construction of Query Donor Strains 3.1.1 Generation of the Linear DNA Fragment for Subsequent Nonessential Gene Deletion Via Recombineering 3.1.2 Amplifying a SPA-Tagging Cassette for Creating a Hypomorphic Allele of an Essential Gene 3.1.3 Preparation of Competent Cells for Donor Mutant Construction 3.1.4 Electroporation and the Selection of Recombineering-Derived Transformants 3.1.5 PCR Confirmation of the Successful Gene Deletion 3.1.6 Confirming Essential Gene Hypomorphic Mutation 3.1.7 Storage of Confirmed Query Mutant Donor Strains Prior to Screening 3.2 Arraying E. coli F- Recipient Strain Collection for Genome-Wide eSGA Screens 3.3 Construction of E. coli Double Mutants Using an Arrayed Strain Mating Procedure 3.4 Data Processing and Score Generation 3.4.1 Quantitative Plate Image Analysis and Colony Size Normalization 3.4.2 Generation of Genetic Interaction Scores 3.4.3 Assessing Genetic Interaction Scores 3.5 Discerning Pathway-Level Relationships 4 Notes References Chapter 2: Mapping Synthetic Dosage Lethal Genetic Interactions in Saccharomyces cerevisiae 1 Introduction 2 Materials 2.1 Drug Stock Solutions 2.2 Media Solutions 2.3 Yeast Strains and Plasmids 2.4 Robotic Pinning System 2.5 Software 3 Methods 3.1 Query Strain Construction 3.2 Mutant Array Construction 3.3 SDL Procedure 3.4 Confirmation of SDL Interactions 4 Notes References Chapter 3: Systematic High-Content Screening of Fluorescently Tagged Yeast Double Mutant Strains 1 Introduction 2 Materials 2.1 Yeast Query Strains and Strain Collections 2.2 Media and Stock Solutions 2.2.1 General Stock Solutions 2.2.2 Media for Synthetic Genetic Array Protocol 2.2.3 Media for High-Throughput Imaging 2.3 Accessories and Equipment 2.3.1 SGA 2.3.2 HTP Imaging 3 Methods 3.1 High-Throughput Fluorescently Tagged Mutant Strain Construction Using SGA 3.2 HTP Imaging 3.2.1 Preparing Plasticware and Media (Done Beforehand) 3.2.2 Preparing Overnight Yeast Cultures (Day 1) 3.2.3 Subculturing (Day 2) 3.2.4 HTP Imaging of Nonessential Gene Deletion Mutants and ORF-GFP Mutants (Day 3) 3.2.5 HTP Imaging of Essential Gene TS Mutants (Day 3) 3.3 Image Analysis and Genetic Interaction Mapping Using HCS Data 4 Notes References Chapter 4: A Genetic Interaction Screening Approach in C. elegans 1 Introduction 2 Materials 2.1 Cloning of sgRNA and Homology Arm Vectors 2.2 Microinjection of C. elegans for CRISPR/Cas9 Genome Editing and Screening Animals 2.3 Relative Fitness Assays 3 Methods 3.1 Selection of Two Single Guide RNAs (sgRNAs) in the Vicinity of Deletion Breakpoints for Given Genes 3.2 Selection of Homology Arms to Generate Partial or Complete Gene Deletions 3.3 Designing sgRNA and Homology Arm Primers 3.4 Creating New sgRNA Vectors by PCR and Ligation 3.5 Amplification of Homology Arms from Genomic DNA 3.6 Digestion of Dual Marker Selection Vectors 3.7 Gibson Assembly of Homology Arms with Digested Dual Marker Selection Vectors 3.8 Preparing CRISPR/Cas9 Injection Mixes and Microinjection 3.9 Antibiotic Selection and Screening of Animals with Gene Deletions 3.10 Establishing Libraries of Double Mutant Animals by Marker Screening 3.11 Phenotyping Animals with a Relative Fitness Assay 4 Notes References Chapter 5: RNA Interference (RNAi) Screening in Cultured Drosophila Cells 1 Introduction 2 Materials 2.1 M3 Insect Media 2.2 Cell Culture 2.3 Polymerase Chain Reaction (PCR) of Complementary DNA (cDNA) 2.4 In Vitro Transcription of Double Stranded RNA (dsRNA) 2.5 dsRNA Quality Control 2.6 dsRNA Treatments 2.7 Sodium Arsenite Treatment 2.8 Immunofluorescent Staining 2.9 RNA Extraction 2.10 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) 2.11 Western Blotting 3 Methods 3.1 Production of dsRNA 3.1.1 PCR Amplification of cDNA Template 3.1.2 In Vitro Transcription of dsRNA 3.2 Cell Culture 3.3 dsRNA Treatment/ RNAi (Cell Bathing Approach) 3.4 Combining dsRNA and Cellular Stress Treatments 3.5 Immunofluorescent Staining of Cells 3.6 High-Content Screening (HCS) Microscopy 3.7 Phenotypic Validations 3.7.1 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) 3.7.2 Western Blotting Analysis 4 Notes References Part II: Approaches to Map Genetic Interactions in Human Cells Chapter 6: Employing Cross-Species Approaches to Construct Humanized Genetic Interaction Networks 1 Introduction 2 Materials 3 Methods 3.1 Accessing Budding Yeast SL Interaction Datasets 3.1.1 Identifying the Budding Yeast Functional Ortholog of the Human Gene of Interest 3.1.2 Accessing Genetic Interaction Data from the BioGRID Database 3.2 Preparing and Filtering BioGRID Interactions Datasets 3.2.1 Selecting SL Interactions 3.2.2 Filter Genetic Interactions Identified in Nonstandard Growth Condition 3.2.3 Filtering Reciprocal Genetic Interactions 3.2.4 Retrieving UniProtKB Unique Entry Accession Numbers 3.3 Prioritizing a Subset of Yeast SL Interactors 3.3.1 Identifying Genetic Interactors Involved in a Biological Process of Interest 3.3.2 Comparing SL Interactions Scores 3.3.3 Prioritizing Pan SL Interactions 3.3.4 Reproducibility of SL Interactions Across Experimental Systems 3.4 Identifying Human Candidate hYFG SL Interactors 3.5 Building a Network of Candidate Human SL Interactors 3.5.1 Generating a Testable Network of Human Candidate SL Interactions 3.5.2 Visualizing the Network of Candidate hYFG SL Interactors 4 Notes References Chapter 7: Identification of Synthetic Lethal Interactions Using High-Throughput, Arrayed CRISPR/Cas9-Based Platforms 1 Introduction 2 Materials 2.1 DNA Isolation 2.2 Cell Culture Reagents 2.3 Cellular Transduction and SLI Testing 3 Methods 3.1 DNA Isolation (Fig. 2) 3.1.1 Cherry-Picking and Bacterial Culture Preparation 3.1.2 High-Throughput DNA Extraction Procedure 3.2 Cas9 Stable Cell Line Generation 3.3 High-hroughput Lentivirus Production (Fig. 3) 3.4 High-hroughput Lentiviral Transduction 4 Notes References Chapter 8: Exploring Candidate Human Synthetic Lethal Interactions Through siRNA and Quantitative Imaging-Based Approaches 1 Introduction 2 Materials 2.1 Cell Culture 2.2 SL Assays 3 Methods 3.1 siRNA-Based Cosilencing Screen of Candidate hYFG SL Interactors 3.1.1 Overarching Principle 3.1.2 Experimental Optimization and Planning 3.1.3 Execution of Cosilencing SL Screen 3.2 siRNA-Based SL Screen in hYFG-Proficient and hYFG-Deficient Isogenic Cell Lines 3.2.1 Experimental Optimization and Preparation 3.2.2 Execution of SL Screens Using Isogenic Cell Line Models 3.3 Prioritization and Validation of Putative SL Interactors 3.3.1 Experimental Validation Is Essential to Identify Bona Fide SL Interactions in Humans 3.3.2 Prioritizing SL Interactors for Experimental Validation 3.3.3 Confirming Gene Silencing with Individual siRNA Duplexes 3.3.4 Assessing SL Phenotypes Associated with Individual siRNA Duplexes 3.3.5 Pursuing Validation 4 Notes References Chapter 9: Mapping Genetic Interactions in Human Cancer Cells Using a One-Step tRNA-CRISPR System 1 Introduction 2 Materials 2.1 Library Construction 2.2 Lentivirus Production and Titration 2.3 Functional Screening and Next Generation Sequencing (NGS) 3 Methods 3.1 Library Construction and Cloning 3.1.1 Design of Pairwise gRNA Constructs 3.1.2 Amplification of the Oligonucleotides 3.1.3 Generation of the Full-Length Segments by Overlapping PCR (Fig. 2) 3.1.4 BsmBI Digestion and Subcloning (Fig. 2) 3.1.5 Transformation and Plasmid Purification (Fig. 2) 3.1.6 Library Lentivirus Production and Titration (Fig. 2) 3.2 Functional Genetic Screening (Fig. 2) 3.2.1 Establishment of HEK293-eSpCas9-Blast Cells 3.2.2 Titration of the Library Virus 3.2.3 Differential Growth Screening (Fig. 2) 3.3 Next-Generation Sequencing (NGS) and Data Analysis (Fig. 2) 3.3.1 Library Preparation and Sequencing 3.3.2 Processing of Paired-End Reads 3.3.3 Data Analysis and Genetic-Interaction Mapping 4 Notes References Chapter 10: In Vivo Genome-Wide Pooled RNAi Screens in Cancer Cells to Identify Determinants of Chemotherapy/Drug Response 1 Introduction 2 Materials 3 Methods 3.1 Selection of shRNA Screening Library 3.2 Optimization of Transfection and Drug Treatment 3.3 In Vivo Selective Pressure 3.4 Deconvolution of Relative shRNA Abundances 4 Notes References Part III: Predicting Genetic Interactions Using Computational Approaches Chapter 11: INCISOR: An Algorithm to Identify Synthetic Rescue Mediators of Resistance to Targeted and Immunotherapy 1 Introduction 2 Materials 2.1 Environment 2.2 Input Requirement 2.3 INCISOR Installation 3 Methods 3.1 In Vitro Screening 3.2 Molecular Survival of the Fittest Screen (SoF) 3.3 Clinical Screening 3.4 Phylogenetic Screening 3.5 Running INCISOR Pipeline 4 Discussion and Conclusion 5 Notes References Chapter 12: Machine Learning to Identify Gene Interactions from High-Throughput Mutant Crosses 1 Introduction 2 Scoring of GIs Using GP Based Model 3 Discussion and Conclusions References Part IV: Mapping Chemical Genetic Interactions Chapter 13: Identification of Drug Resistance Genes Using a Pooled Lentiviral CRISPR/Cas9 Screening Approach 1 Introduction 2 Materials 2.1 sgRNA Viral Library 2.2 Mammalian Cell Culture 2.3 Screening 3 Methods 3.1 Selection Antibiotic Kill Curve 3.2 Drug/Ligand Kill Curve 3.3 Determination of Viral Multiplicity of Infection (MOI) 3.4 Preparation of Transduced Library Cells 3.5 Drug Resistance Screening 3.6 Genomic DNA Extraction 3.7 sgRNA Amplification 3.8 PCR Product Gel Purification 3.9 Next Generation Sequencing 3.10 Data Analysis 4 Notes References Chapter 14: Chemical-Genetic Interactions as a Means to Characterize Drug Synergy Abbreviations 1 Introduction 2 A Chemogenomics Primer 2.1 Forward, Reverse, and Predictive Chemogenomics 2.2 Gene-Gene Interactions 2.3 Gene-Drug Interactions 2.4 Haploinsufficiency Profiling (HIP) 2.5 Homozygous Profiling (HOP) 2.6 Gene-Drug-Drug Interactions 2.7 Model Organisms in Chemogenomics 3 Synergy from an Experimental Perspective 3.1 Drug Synergy from an Experimental Perspective 3.2 Case Study Examples 4 Summary and Perspective References Part V: Emerging Technologies That Leverage Mapping Genetic Interactions Chapter 15: Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryo... 1 Introduction 2 Materials 2.1 Drosophila Maintenance and Embryo Collection 2.2 MCF7 Tissue Culture 2.3 Puro-PLA and Immunofluorescence 3 Methods 3.1 Harvesting and Treating DrosophilaEmbryos for Puro-PLA 3.1.1 Embryo Collection 3.1.2 Embryo Dechorionation 3.1.3 Translation Inhibitor Treatment 3.1.4 Fixation 3.1.5 Vitelline Permeabilization and Removal 3.2 Culturing and Treating Cells for Puro-PLA 3.2.1 Culturing Cells 3.2.2 Translation Inhibitor Treatment 3.2.3 Fixation 3.3 Immunolabeling and PLA-Oligos Application in DrosophilaEmbryos and Cultured Cells 3.3.1 Primary Antibodies Labeling Drosophila Cells 3.3.2 Secondary Labeling with Complementary Probes Drosophila Cells 3.3.3 Ligation Drosophila Cells 3.3.4 Circle-Forming Amplification and Binding Probes Drosophila Cell 3.4 Dual Labeling with Immunofluorescence 3.4.1 Demarcating Region with Marker 3.5 Revelation of Targeted Puro-PLA Signal 3.5.1 Staining DNA and Signal Revelation Drosophila Cells 4 Notes References Chapter 16: Inferring Copy Number from Triple-Negative Breast Cancer Patient Derived Xenograft scRNAseq Data Using scCNA 1 Introduction 2 Materials 2.1 Media and Stock Solutions 2.2 Dissociation Accessories and Kits 3 Methods 3.1 Automated Isolation of TNBC PDX and Normal Breast Epithelial Single-Cell Suspension 3.2 Manual Isolation of TNBC PDX and Normal Breast Epithelial Single-Cell Suspension 3.3 Single-Cell RNA Sequencing Using Droplet-Based Technology from 10x Genomics 3.3.1 Cell Preparation 3.3.2 Prepare RT Master Mix 3.3.3 Load the Chromium Single-Cell 3′ Chip and GEM Harvest 3.3.4 GEM RT, Cleanup, and cDNA Amplification 3.3.5 Amplified cDNA Clean Up 3.3.6 Sequence Ready Library Construction: cDNA Fragmentation, End Repair, A-Tailing 3.3.7 Sequence Ready Library Construction: Adaptor Ligation 3.3.8 Sequence Ready Library Construction: Sample Index PCR Mix 3.3.9 scCNA: Single-Cell RNA-Seq Data Analysis for Identifying Copy Number Aberrations 4 Notes References Part VI: Downstream Validation of Genetic Interactions Using Protein Inhibitors and Small Molecules Chapter 17: Generation of Protein Inhibitors for Validation of Cancer Drug Targets Identified in Functional Genomic Screens 1 Introduction 2 Materials 2.1 Stock Solutions 2.2 Commercial Kits, Enzymes, and Reagents 2.3 Buffers, Solutions, and Media 2.4 Bacterial and Phage Strains 2.5 Plasmids and Oligonucleotides 2.6 Specialized Equipment 3 Methods 3.1 Phagemid Design 3.2 Library Design 3.3 Library Construction 3.3.1 Purification of dU-ssDNA 3.3.2 Synthesis and Purification of Heteroduplex ccc-dsDNA 3.3.3 Preparation of Preinfected Electrocompetent SS320 Cell Stock and M13K07 Lab Stock 3.3.4 Electroporation of Heteroduplex DNA and Library Preparation 3.4 Expression of Recombinant Target Proteins 3.4.1 Expression of Recombinant Proteins and Preparation of Lysates 3.4.2 Empirical Optimization of Recombinant Protein Immobilization on ELISA Plates 3.5 Phage Library Screening for Selecting Binding Proteins 3.6 Phage Pool ELISA and Analysis of Single-Phage Clones 3.6.1 Phage Pool ELISA 3.6.2 Isolating and Analyzing Individual Clones 3.6.3 Sanger Sequencing to Identify Specific Binders 3.6.4 Verifying Inhibition of Target Proteins in Cancer Cells 4 Notes References Chapter 18: Computational Prediction of Chemical Tools for Identification and Validation of Synthetic Lethal Interaction Netwo... 1 Introduction 2 Publicly Available Bioactivity Resources 2.1 ChEMBL (https://www.ebi.ac.uk/chembl/) 2.2 PubChem (http://pubchem.ncbi.nlm.nih.gov/sources#assay) 2.3 BindingDB (https://www.bindingdb.org) 2.4 DrugBank (https://www.drugbank.ca/) 3 Commercial Libraries of Small Molecules 4 Protein Data Bank (https://www.rcsb.org/) 5 Homology Modeling and Validation 6 Virtual Screening 6.1 Molecular Docking 6.1.1 DOCK 6.1.2 AutoDock 6.1.3 Glide 6.1.4 FlexX 6.1.5 GOLD 6.1.6 Miscellaneous 6.2 Postdock Processing 6.2.1 Improving Computational Efficiency with Hierarchical Screening 6.3 Prediction of Ligand Selectivity and Specificity 6.3.1 Computational Study of Ligand Selectivity Remains a Great Challenge 6.4 De Novo Drug Design 7 Conclusions 7.1 Current Challenges and Potential Solutions 7.2 Future Directions References Index