Essential Genes and Genomes: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9
	1 Introduction
	2 Materials
		2.1 CRISPR gRNA Library
			2.1.1 gRNA Library Selection and Amplification
			2.1.2 gRNA Library Lentivirus Production
		2.2 Screening Cell Line
			2.2.1 Cell Line Selection
			2.2.2 Generation of Stable, Inducible Cas9-Expressing Cell Line
			2.2.3 Prescreen Optimization, Cell Line Characterization and Quality Control Procedures
		2.3 CRISPR Screen
			2.3.1 Determination of Lentivirus Multiplicity of Infection (MOI)
			2.3.2 gRNA Library Transduction, Cell Passaging, Sampling and Quality Control
			2.3.3 Genomic DNA Isolation
			2.3.4 Sequencing Library Preparation
			2.3.5 Next-Generation Sequencing
			2.3.6 Data Analysis
	3 Methods
		3.1 CRISPR gRNA Library
			3.1.1 gRNA Library Selection
			3.1.2 gRNA Library Amplification
			3.1.3 gRNA Library Lentivirus Production
		3.2 Screening Cell Line
			3.2.1 Cell Line Selection
			3.2.2 Generation of Stable, Inducible Cas9-Expressing Cell Line
			3.2.3 Prescreen Optimization, Cell Line Characterization and Quality Control Procedures
		3.3 CRISPR Screen
			3.3.1 Determination of Lentivirus Multiplicity of Infection (MOI)
			3.3.2 gRNA Library Transduction, Cell Passaging, Sampling and Quality Control
			3.3.3 Genomic DNA Isolation
			3.3.4 Sequencing Library Preparation
			3.3.5 Next-Generation Sequencing
			3.3.6 Data Analysis
	4 Notes
	References
Chapter 2: CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines
	1 Introduction
	2 Materials
		2.1 Viral Packaging
		2.2 Titer  Test
		2.3 Screen Infection
		2.4 Screen Cell Culture and Library Preparation
	3 Methods
		3.1 Viral Packaging
		3.2 Titer  Test
		3.3 Screen Infection
		3.4 Screen Cell Culture and Library Preparation
		3.5 Data Analysis
	4 Notes
	References
Chapter 3: Target Identification of Small Molecules Using Large-Scale CRISPR-Cas Mutagenesis Scanning of Essential Genes
	1 Introduction
	2 Materials
		2.1 sgRNA Library Cloning
		2.2 Lentivector Production
		2.3 Lentivector Titration
		2.4 Stable SpCas9 Cell Line Generation
		2.5 CRISPR-SpCas9 Compound Screen
		2.6 DNA Library Preparation
		2.7 Validation of Individual  Hits
	3 Methods
		3.1 In Silico sgRNA Tiling Library Design
		3.2 sgRNA Library Cloning
		3.3 Lentivector Production
		3.4 Lentivector Titration
		3.5 Stable SpCas9 Cell Line Generation
		3.6 CRISPR-SpCas9 Compound Screen
		3.7 DNA Library Preparation
		3.8 Next-Generation Sequencing of sgRNAs
		3.9 Validation of Individual  Hits
		3.10 Sample Preparation and Next-Generation Sequencing of Target Site Variants
	4 Notes
	References
Chapter 4: Design and Generation of a CRISPR Interference System for Genetic Repression and Essential Gene Analysis in the Fun...
	1 Introduction
	2 Materials
		2.1 Duplexing of Forward and Reverse Oligonucleotides for the sgRNA
		2.2 Plasmid Extraction
		2.3 Golden Gate Cloning of sgRNA
		2.4 Transformation into Competent E. coli Cells
		2.5 PCR Verification
		2.6 Archiving Plasmids
		2.7 Linearization of the Cloned Plasmid
		2.8 Transformation of Cloned Plasmids into C. albicans
		2.9 Verification of Integration into C. albicans
		2.10 qRT-PCR to Confirm Repression of Gene of Interest
	3 Methods
		3.1 Design of sgRNA
		3.2 Duplexing of Forward and Reverse Oligos for the sgRNA
		3.3 Plasmid Extraction from Bacterial Culture
		3.4 Golden Gate Cloning of sgRNA into CRISPRi Plasmid Backbone
		3.5 Transformation into Competent E. coli Cells
		3.6 PCR Verification for Cloned Plasmids
		3.7 Archiving Plasmids for Further  Use
		3.8 Linearization of Cloned Plasmids
		3.9 Transformation of Cloned Plasmids into C. albicans
		3.10 Verification of Integration into C. albicans Genome
		3.11 qRT-PCR to Confirm Repression of Gene of Interest
	4 Notes
	References
Chapter 5: Identification of Essential Genes Using Sequential CRISPR and siRNA Screens
	1 Introduction
	2 Materials
		2.1 Genome-Wide Crispr Screen
		2.2 Library Preparation
		2.3 Lentivirus Production
		2.4 Determination of Infection Efficiency
		2.5 Library Transduction
		2.6 Genomic DNA Extraction
		2.7 PCR and Purification for Sequencing
		2.8 Pooled siRNA Validation Screen
		2.9 Deconvolution Screen
	3 Methods
		3.1 Library Preparation
			3.1.1 Preparation of LB Agar Plates
			3.1.2 Electroporation and Bacterial Growth
			3.1.3 Plasmid DNA Extraction
		3.2 Lentivirus Production
			3.2.1 Day 1: Cell Seeding
			3.2.2 Day 2: 293T Cell Preparation for Transfection
			3.2.3 Day 2: Transfection Master Mix Formulation
			3.2.4 Day 2: 293T Plasmid DNA Transfection
			3.2.5 Day 4: Virus Media Collection and Purification
		3.3 Determination of Infection Efficiency
			3.3.1 Before Starting Protocol
			3.3.2 Day 1-Cell Seeding
			3.3.3 Day 2-Viral Transduction
			3.3.4 Day 3-Drug Selection
			3.3.5 Day 6-Calculation of Infection Efficiency
		3.4 Library Transduction
			3.4.1 Required Calculations
			3.4.2 Day 1-Seeding T-225 Flasks
			3.4.3 Day 2-Library Lentivirus Transduction
			3.4.4 Day 3-Puromycin Selection
			3.4.5 Day 6-11-Cell Maintenance
		3.5 Dropout Screen
			3.5.1 Day 0-Maintain gRNA Library Diversity for 8-18 Doublings
		3.6 Genomic DNA Extraction
		3.7 PCR and Purification for Sequencing
			3.7.1 PCR Round 1 (Nested PCR)
			3.7.2 PCR Round 2
			3.7.3 Purification of PCR Products
		3.8 Illumina Sequencing and Data Analysis
		3.9 Validation Using siRNA Screening
			3.9.1 Pooled siRNA Validation Screen
			3.9.2 Deconvolution Screen
	4 Notes
	References
Chapter 6: A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Vario...
	1 Introduction
	2 Materials
		2.1 Plasmids
		2.2 Transfection
	3 Methods
		3.1 Plasmid Construction
			3.1.1 pX330-Based Plasmid Construction
			3.1.2 pAID Plasmid Construction
		3.2 Transfection of Plasmids
			3.2.1 Transfection into a Chicken DT40 Cell  Line
			3.2.2 Transfection into Human Cell Lines
			3.2.3 Transfection into Mouse ES Cell Line (E14tg2a)
	4 Notes
	References
Chapter 7: New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Scr...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Construction and Characterization of CRISPRi System in E. coli
		3.2 Design and Preparation of Genome-Wide sgRNA Library
			3.2.1 Design Process
			3.2.2 Preparation of sgRNA Library
		3.3 Pooled Screening Experiments
			3.3.1 Preparation of E. coli Electroporation-Competent Cells
			3.3.2 Plasmids Transformation
			3.3.3 sgRNA Library Screening
		3.4 NGS Library Preparation and Sequencing
			3.4.1 PCR Amplification
			3.4.2 Library Construction and Sequencing
		3.5 NGS Data Processing
			3.5.1 Read Mapping
			3.5.2 Calculation of sgRNA Fitness
			3.5.3 Calculation of Gene Fitness
		3.6 ROC-AUC to Compare Performance of Different Methods for Identification of Essential Genes
		3.7 Software Tools
	4 Notes
	References
Chapter 8: A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria
	1 Introduction
	2 Materials
		2.1 Strains, Plasmids, and Primers
		2.2 Media, Antibiotics, and Other Additives
		2.3 Molecular Biology Reagents and Kits
		2.4 Equipment, Glassware, and Disposable Materials
	3 Methods
		3.1 Design and Construction of the Genome Editing Plasmid
		3.2 Conjugation
		3.3 Genotype and Phenotype Verification
		3.4 Curing the Mutant Cells of the Editing Plasmid
	4 Notes
	References
Chapter 9: A Method of Transposon Insertion Sequencing in Comprehensively Identifying Vibrio vulnificus Genes Required for Gro...
	1 Introduction
		1.1 Transposon Insertion Sequencing
		1.2 Microorganism Model: The Septicemic Bacterium Vibrio vulnificus
	2 Materials
	3 Methods
		3.1 Strain Selection
		3.2 Library Preparation (Fig. 2)
		3.3 Selection of Transconjugants (Fig. 2)
		3.4 DNA Extraction
		3.5 Sibling Test
		3.6 Library Selection (Fig. 3)
		3.7 Sequencing (Fig. 4)
		3.8 Analysis of Transposon Insertion Sites. Under-Represented Genes Detection (Fig. 5)
		3.9 Bioinformatic Analyses
	4 Notes
	References
Chapter 10: The Use of Tn-Seq and the FiTnEss Analysis to Define the Core Essential Genome of Pseudomonas aeruginosa
	1 Introduction
	2 Materials
		2.1 Plasmids, Strains, Antibiotics, and Agar Selection Media
		2.2 Illumina Library Preparation and Sequencing
		2.3 Analysis Requirements
	3 Methods
		3.1 Generating Transposon Mutant Libraries
			3.1.1 Day 1
			3.1.2 Day 2
			3.1.3 Day 3
			3.1.4 Day 4
			3.1.5 Days 5-6
		3.2 Preparing DNA Libraries for Illumina Sequencing
		3.3 Data Analysis and Identifying Essential and Nonessential Genes
			3.3.1 Preparing your Computer and Files for Analysis
			3.3.2 Identifying and Filtering TA Insertion Sites from the Genomic Sequence
			3.3.3 Mapping Sample Data to the Genome and Tallying Mapped Reads
			3.3.4 Using FiTnEss to Identify Essential and Nonessential Genes
	4 Notes
	References
Chapter 11: Analysis of Escherichia coli K1 Virulence Genes by Transposon-Directed Sequencing
	1 Introduction
	2 Materials
		2.1 Preparation of Electrocompetent E. coli
		2.2 Transformation of E. coli with EZ-Tn5 Transposome
		2.3 Harvesting and Pooling E. coli Tn5 Mutants
		2.4 Counting Colony-Forming Units in E. coli Tn5  Pool
		2.5 Extracting DNA from E. coli Tn5 Library
		2.6 Fragmentation of  DNA
		2.7 Repairing the Ends of Fragmented  DNA
		2.8 Addition of A-Tail to Repaired DNA Fragments
		2.9 Ligation of Adapters to DNA Fragments
		2.10 PCR Amplification of Tn Insertion Sites
		2.11 Purification of PCR Amplicons
		2.12 Sample Indexing
		2.13 Clean-up Size Indexed Fragments
		2.14 Sequencing on MiSeq
	3 Methods
		3.1 Preparation of Electrocompetent E. coli
		3.2 Transformation of E. coli with EZ-Tn5 Transposome
		3.3 Harvesting and Pooling E. coli Tn5 Mutants
		3.4 Counting Colony-Forming Units in E. coli Tn5  Pool
		3.5 Extracting DNA from E. coli Tn5 Library
		3.6 Fragmentation of  DNA
		3.7 Repairing the Ends of Fragmented  DNA
		3.8 Addition of A-Tail to Repaired DNA Fragments
		3.9 Ligation of Adapters to DNA Fragments
		3.10 PCR Amplification of Tn Insertion Sites
		3.11 Purification of PCR Amplicons
		3.12 Sample Indexing
		3.13 Clean-up Size Indexed Fragments
		3.14 Sequencing of Amplicons with MiSeq and Processing of Sequence Reads
	4 Notes
	References
Chapter 12: Using Genome Scale Mutant Libraries to Identify Essential Genes
	1 Introduction
	2 Materials
		2.1 Growing Bacteria
		2.2 Tn-seq Sample Preparation
		2.3 Tn-seq Data Analysis
	3 Methods
		3.1 Mutant Library Construction
		3.2 Transposon Library Propagation
		3.3 Tn-seq Sample Preparation
		3.4 DNA (Illumina) Sequencing
		3.5 Tn-seq Data Analysis
	4 Notes
	References
Chapter 13: Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing
	1 Introduction
	2 Materials
		2.1 In Vitro Transposition
		2.2 Transformation of S. mutans Transposon Library
		2.3 In Vitro Library Selection
		2.4 Genomic DNA Isolation
		2.5 Transposon Sequencing Library Preparation
		2.6 Transposon DNA Library Sequencing
		2.7 Data Analysis
	3 Methods
		3.1 In Vitro Transposition of S. mutans Genomic  DNA
		3.2 Transformation of S. mutans Transposon Library
		3.3 Library Selection
		3.4 Genomic DNA Isolation
		3.5 Transposon Sequencing Library Preparation
		3.6 Transposon DNA Library Sequencing
		3.7 Data Analysis
	4 Notes
	References
Chapter 14: The Use of TnSeq to Identify Essential Alphaproteobacterial Genes Reveals Operational Variability in Conserved Dev...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Transposon Libraries
		3.2 DNA Extraction, Preparation, and Sequencing
		3.3 Data Analysis
	4 Notes
	References
Chapter 15: A Proposed Framework to Identify Dispensable and Essential Functions in Bifidobacteria: Case Study of Bifidobacter...
	1 Introduction
	2 Materials
		2.1 Basic Microbiology Reagents
		2.2 Basic Molecular Biology Reagents
		2.3 Equipment
	3 Methods
		3.1 Transposon Mutagenesis Strategy for B. breve UCC2003
			3.1.1 Transposon Construction and Preparation
			3.1.2 Preparation of Electrocompetent Cells of Bifidobacterium (See Note 7) and Generation of a Collection of Transposon Inser...
		3.2 TraDIS Sequencing Library Preparation
			3.2.1 Total DNA Isolation
			3.2.2 Manual TraDIS Library Protocol
		3.3 Approaches to Essential Gene Prediction in Bifidobacteria from Datasets Generated Through TIS Approaches
			3.3.1 Sequencing and Quality Control
			3.3.2 Essential Gene Prediction Workflow Applied to TIS Experimental Approaches in Bifidobacteria
		3.4 Comparative Analysis of Essential Gene and Core Genome Functions Across Bacteria
	4 Notes
	References
Chapter 16: The Application of Transposon Insertion Sequencing in Identifying Essential Genes in B. fragilis
	1 Introduction
	2 Materials
		2.1 Transposon Mutant Library (TML) Generation
		2.2 Sequencing Library Preparation and Sequencing
			2.2.1 Genomic DNA (gDNA) Isolation, gDNA Shearing, and Adding C-Tail
			2.2.2 Transposon Insertion Site (TIS) Amplification
			2.2.3 Sample Barcoding PCR, Sequencing, and Analysis
	3 Methods
		3.1 Transposon Mutant Library (TML) Generation
		3.2 Sequencing Library Preparation and Sequencing
			3.2.1 Genomic DNA (gDNA) Isolation, gDNA Shearing, and Adding C-Tail
			3.2.2 Transposon Insertion Site (TIS) Amplification
			3.2.3 Sample Barcoding PCR, Sequencing, and Analysis
	4 Notes
	References
Chapter 17: Repressible Promoter System to Study Essential Genes in Mycobacteria
	1 Introduction
		1.1 Strategies to Generate Conditional Mutants
			1.1.1 Selection of the Promoter
			1.1.2 Construction of the Conditional Mutant (First Approach: Promoter Replacement)
			1.1.3 Construction of the Conditional Mutant (Second Approach: Ectopic Expression)
		1.2 Working Conditions
		1.3 Applications
	2 Materials
		2.1 Cloning and Manipulation Procedures in Escherichia coli
		2.2 Mycobacterial Culture Media
		2.3 Mycobacterial DNA Extraction
		2.4 Generation of Single-Stranded DNA by Alkali Denaturation
		2.5 Electroporation of Mycobacteria
		2.6 Fluorescence Assay
		2.7 Plasmids
			2.7.1 pFRA50
			2.7.2 pFRA50-Derivative Plasmids
			2.7.3 pFRA61
		2.8 Stock Solutions of Inducers
		2.9 Infection of Macrophages
		2.10 Mice Infections
	3 Methods
		3.1 Construction of a Reporter Plasmid to Evaluate the Strength of the Promoter of the Gene of Interest
		3.2 Fluorescence Assay
		3.3 Construction of Conditional Mutants (First Approach, Fig. 3)
			3.3.1 Preparation of Suicide Vector
			3.3.2 Mutant Selection
			3.3.3 Introduction of the TetR/Pip OFF System
		3.4 Construction of Conditional Mutants (Second Approach, Fig. 4)
		3.5 Preparation of Single-Stranded Denatured Plasmid  DNA
		3.6 Electroporation
		3.7 Validation of Gene Essentiality with TetR/PipOFF System In Vitro
		3.8 Infection of Macrophages
		3.9 Mice Infection
	4 Notes
	References
Chapter 18: A Single-Cell RNA-Seq-Based Approach for Genome-Wide Identification of Cell Essential Genes
	1 Introduction
	2 Materials
	3 Methods
		3.1 Isolation of Mouse Glomeruli Using Dynabeads and Magnetic Concentrator
		3.2 Preparation of Mouse Glomerular Single-Cell Suspension
		3.3 Single-Cell Separation and Preparation of cDNA Samples
		3.4 Quality Assessment of cDNA Samples and Identification of Cell Type of  cDNA
		3.5 Construction of cDNA Library for Sequencing
		3.6 Sequencing
		3.7 Identification of Podocyte Essential Gene Candidates Based on the Single-Podocyte Gene Expression Profiles
		3.8 Assessment of Essentiality of the 92 Podocyte-Specific Essential Gene Candidates
	4 Notes
	References
Chapter 19: Identification of Essential Genes in Caenorhabditis elegans with Lethal Mutations Maintained by Genetic Balancers
	1 Introduction
	2 Materials
		2.1 Strain Maintenance and Sequencing
			2.1.1 The Maintenance of Each C. elegans Strain
			2.1.2 Genomic DNA Extraction and Sequencing
		2.2 Database Used in the Protocol
		2.3 Software Used in the Protocol
	3 Methods
		3.1 Maintaining C. elegans Strains and Whole-Genome Sequencing
			3.1.1 Maintaining C. elegans Strains Carrying Lethal Mutations
			3.1.2 Genomic DNA Extraction and Sequencing
		3.2 Mutation Identification
			3.2.1 Download the Reference Genome and the Sequencing Data
			3.2.2 Aligning the FASTQ Reads to the C. elegans Reference Genome
			3.2.3 Realignment Around Indels
			3.2.4 Variation Calling
			3.2.5 Identify High Confidence Variations
			3.2.6 Identify the High-Confidence Candidate Essential Genes
			3.2.7 Confirmation Candidate Essential Gene by PCR of Second Alleles
		3.3 Confirmation by the Complementation Testing
	4 Notes
	References
Chapter 20: A Screening Method to Identify Essential Yeast Genes for Responses Towards Spermine
	1 Introduction
	2 Materials
		2.1 Strains
		2.2 Media
		2.3 Drugs
		2.4 Equipment and Supplies
	3 Methods
		3.1 Inoculation, Adjustments of Cell Density, and Dilution of the Cells for Spot  Test
		3.2 Preparation of Spermine-Containing Plates
		3.3 Spot Test Assay
		3.4 Uptake Assay
		3.5 Data Analyses
	4 Notes
	References
Chapter 21: An Effective Preprocessing Method for High-Quality Pan-Genome Analysis of Bacillus subtilis and Escherichia coli
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sequencing Genome  Data
		3.2 Genome Annotation
		3.3 Phylogenetic Relationship Analysis
		3.4 Average Nucleotide Identity Analysis
		3.5 Pan-Genome Analysis
		3.6 Gene Functional Enrichment Analysis
		3.7 Pan-Genome State
		3.8 Pan-Genome of E. coli
	4 Notes
	References
Chapter 22: Analysis of Gene Essentiality from TnSeq Data Using Transit
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preprocessing (TPP)
			3.1.1 Making Combined Wig Files
			3.1.2 Evaluating Quality of TnSeq Data
		3.2 Analyses for Single Conditions
			3.2.1 Gumbel Analysis
			3.2.2 Prot Tables
			3.2.3 Hidden Markov Model
		3.3 Pairwise Comparisons: Resampling
			3.3.1 Pathway Analysis
		3.4 Analyses of Multiple Conditions
			3.4.1 Genetic Interaction Analysis
			3.4.2 ANOVA
			3.4.3 Zero-Inflated Negative Binomial (ZINB)
	4 Summary
	5 Notes
	References
Chapter 23: Geptop 2.0: Accurately Select Essential Genes from the List of Protein-Coding Genes in Prokaryotic Genomes
	1 Introduction
	2 Materials
		2.1 Reference Genomes and Cross-Species Validation
		2.2 Introduction of Web  Page
	3 Methods
		3.1 Input and Output of Geptop
		3.2 Usage of Online Service
		3.3 Usage of Standalone Version
	4 Additional Noted Information for users of Geptop
	5 Conclusion
	References
Index