Epigenome Editing: Methods and Protocols (Methods in Molecular Biology, 2842)

Preface
Contents
Contributors
Part I: Introduction
	Chapter 1: Development of Locus-Directed Editing of the Epigenome from Basic Mechanistic Engineering to First Clinical Applica...
		1 Introduction
		2 History and Development of EpiEditing
		3 Modules Used for EpiEditing
			3.1 DNA Binding Domains (DBD)
			3.2 Functional Domains
		4 Critical Parameters of EpiEditing
			4.1 Specificity of EpiEditing
			4.2 Stability of EpiEditing
		5 Conclusions and Future Perspectives for EpiEditing
		References
Part II: Epigenome Editing Reviews
	Chapter 2: Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities
		1 Introduction
		2 Biochemical Specificity
			2.1 Biological Considerations
				2.1.1 Epigenome Biochemistry Exhibits Complex Natural Diversity
				2.1.2 Epigenome Modifications Exist in Combinations
				2.1.3 Epigenome Modifications Can Be Interdependent
			2.2 Design Considerations and Methods to Assess and Improve the Biochemical Specificity of EEs
				2.2.1 Using Functional Domains Versus Full-Length Proteins to Improve Biochemical Specificity
				2.2.2 Using Multiple Chromatin Modifying Enzymes to Increase Activity
				2.2.3 Advanced Multiplexing Technologies
				2.2.4 Chromatin Immunoprecipitation to Determine Biochemical Specificity
				2.2.5 In Vitro Screening of Biochemical Specificity of CMEs
				2.2.6 Mutations to Reveal Catalytically Important Residues
			2.3 Future  Work
		3 Locus Specificity
			3.1 Biological Considerations
				3.1.1 Off-Target Binding
				3.1.2 Fusion-Dependent Changes of DBD and CME Activities
				3.1.3 DNA Accessibility
			3.2 Design Considerations and Methods to Assess and Improve Locus Specificity
				3.2.1 Binding Affinity and Concentration Effects on Locus Specificity
				3.2.2 Using Obligate Pairs of Chromatin Modifying Enzyme Domains to Improve Locus Specificity
				3.2.3 Using Small Molecules to Recruit Endogenous Proteins to Facilitate Epigenome Editing
				3.2.4 Computational Selection of gRNAs for dCas9 Targeting
				3.2.5 Altering or Removing the Nonspecific Binding Activity of the CME to Enhance Locus Specificity
				3.2.6 Reducing Overall DNA-Binding Energies to Reduce Nonspecific Binding
				3.2.7 Truncated gRNAs Increase Locus Specificity in Cas9 Applications
				3.2.8 Structure Guided Alterations to DBDs to Reduce Nonspecific Binding
				3.2.9 ChIP-Seq to Determine Locus Specificities of EEs
				3.2.10 Mismatch Reporter Assays
				3.2.11 Future Prospects for Epigenome Editors
			3.3 Additional Considerations
		References
	Chapter 3: Fine-Tuning the Epigenetic Landscape: Chemical Modulation of Epigenome Editors
		1 Introduction
		2 Split dCas9-Based Epigenome Editing Systems
			2.1 Inducible Reassembly of Split-dCas9
			2.2 Inducible Tethering of Effector Domains to dCas9
		3 Targeted Degradation of Epi-Editors
			3.1 Epi-Editor Degron Fusions
			3.2 PROTACs
		4 Small Molecule Recruitment of Endogenous Effector Domains
		5 Controlling dCas9 Binding to sgRNA
			5.1 CiCas9
			5.2 Anti-CRISPR/Cas9 Proteins
		6 Conclusions and Perspectives
		References
	Chapter 4: Using High-Throughput Measurements to Identify Principles of Transcriptional and Epigenetic Regulators
		1 Locus-Specific Epigenome Editing and Transcriptional Regulation
		2 Most Commonly Used Locus-Specific, CRISPR-Based Transcriptional Effectors and Epigenetic Editors
			2.1 CRISPRa Activation
			2.2 CRISPRi Repression
		3 HT-Recruit: A High-Throughput Recruitment Assay for Measuring Thousands of Candidate Transcriptional Effector Domains in Par...
		4 Considerations for Effector Domain Library Design
			4.1 Ensuring Reproducibility
			4.2 DNA Binding Domain
			4.3 Combinations of Effector Domains
			4.4 Protein Size
			4.5 Cell Type and Signaling Pathways
		5 Considerations for Understanding Context-Dependent Effects
			5.1 Effects of Local Chromatin Context on Activation and Silencing
			5.2 Effects of Chromatin Context on Memory of Activation and Silencing
		6 Future Work
			6.1 High-Throughput Measurements of Effector Domain Cofactor Dependencies
			6.2 Directions for Tool Improvement
		References
	Chapter 5: Reader-Effectors as Actuators of Epigenome Editing
		1 Introduction: The Role of Reader-Effectors in Epigenome Editing
			1.1 An Overview of Reader-Effectors That Interact with Marks Generated by Popular Epigenome Editors
				1.1.1 DNA Methylation
				1.1.2 Histone H3 Lysine 27 Acetylation
				1.1.3 Histone H3 Lysine 4 Trimethylation
				1.1.4 Histone H3 Lysine 9 Trimethylation
				1.1.5 Histone H3 Lysine 27 Trimethylation
				1.1.6 Histone H3 Lysine 79 Methylation
			1.2 Intrinsic Effects: How Epigenetic Marks Directly Affect Chromatin-Mediated Gene Regulation Across Length Scales
				1.2.1 Transcription Factor Binding Sites
				1.2.2 Single Nucleosome Structure
				1.2.3 Local Chromatin Compaction
				1.2.4 Large-Scale Genome Compaction
			1.3 Extrinsic Activity: Interactions of Reader-Effectors with Chromatin Signals Are Critical for Epigenetic Regulation
		2 Potential Consequences of Disrupted Reader-Effector Activity for Epigenome Editing
			2.1 Inefficient and Unstable Epigenome Editing Outputs
			2.2 Abnormal and Context-Specific Reader-Effector Activity
				2.2.1 DNA Methylation
				2.2.2 Histone H3 Lysine 27 Acetylation
				2.2.3 Histone H3 Lysine 9 Trimethylation
				2.2.4 Histone H3 Lysine 27 Trimethylation
				2.2.5 Histone H3 Lysine 79 Methylation
			2.3 Chromatin Marks That Affect Reader-Effector Binding
		3 How Synthetic Reader-Effectors Can Augment the Effects of Epigenome Editing
			3.1 Synthetic Reader-Actuators That Target DNA Methylation
			3.2 Synthetic Reader-Actuators That Target Histone H3 Lysine 27 Trimethylation
			3.3 Other Epigenome Edits That Are Potentially Compatible with SRAs
		4 Conclusion
		References
	Chapter 6: Neuroepigenetic Editing
		1 Introduction
		2 Epigenetic Editing Tools
		3 Special Considerations for Epigenetic Editing in Neuroscience
			3.1 In Vivo Delivery Methods
			3.2 Spatial and Temporal Control
		4 Applications to Neurological Disorders
			4.1 Neurodegenerative Disorders
			4.2 Neuropsychiatric Disorders
			4.3 Neurodevelopmental Disorders
		5 Applications to Basic Neuroscience
		6 Future Applications: Combinatorial Approaches
		7 Conclusion
		References
Part III: Epigenome Editing Technology
	Chapter 7: Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms
		1 Introduction
		2 Materials
			2.1 Plasmids
			2.2 Preparation of Vector
			2.3 Transfection of Cells
			2.4 Validation of gRNA
			2.5 DNA Methylation Analysis
			2.6 Gene Expression Analysis
		3 Methods
			3.1 Validation of gRNA
				3.1.1 gRNA Design
				3.1.2 Preparation of the Double-Stranded Oligonucleotide
				3.1.3 Preparation of Vector
				3.1.4 Ligation and Transformation
				3.1.5 Validation of gRNA (see Note 2)
			3.2 Transfection for DNA Demethylation at Specific DNA Loci (see Note 3)
			3.3 Transfection for Gene Activation with Recruitment of TET1 and VP64
			3.4 Bisulfite Modification (see Note 6)
			3.5 Bisulfite PCR Amplification
				3.5.1 COBRA
				3.5.2 Amplicon-Based Bisulfite Sequencing (see Note 8)
				3.5.3 Quantitative RT-PCR Analysis
		4 Notes
		References
	Chapter 8: Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects
		1 Introduction
		2 Materials
			2.1 DNA Methylation Editing in Cultured Cells
			2.2 PCR and Purification
			2.3 Quantification of Purified PCR Products
			2.4 Sequencing Using MiSeq
			2.5 Analysis of MiSeq  Data
		3 Methods
			3.1 Transfection and Cell Sampling
			3.2 First PCR for Amplicon-Seq (Bisulfite-PCR, ChIP-PCR, and RT-PCR)
			3.3 Sample Preparation for Amplicon-Seq Using MiSeq
			3.4 Sequencing Using MiSeq
			3.5 Setting of Illumina Experiment Manager, the MiSeq Software (Note 11)
			3.6 Analysis of MiSeq  Data
		4 Notes
		References
	Chapter 9: Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9
		1 Introduction
		2 Materials
			2.1 gRNA Cloning Using Golden Gate Assembly
			2.2 Cell Culture
		3 Methods
			3.1 gRNA Design
			3.2 gRNA Cloning
			3.3 Transfection of the Cells with Epieditors
		4 Notes
		References
	Chapter 10: Plant Epigenetic Editing to Analyze the Function of Histone Modifications in Gene-Specific Regulation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Vector Production
				3.1.1 Selecting and Designing gRNA Cassettes
				3.1.2 Generating gRNA Cassettes
				3.1.3 Vector Preparation for Testing of sgRNAs: gRNA-dCas9-VP64 Vectors
				3.1.4 Generating gRNA-dCas9-VP64
				3.1.5 Testing the sgRNAs by Transient Expression in Tobacco Leaves
				3.1.6 Vector Preparation for Generating Stable Transgenic Lines: Preparation of gRNA-dCas9-Modifier Vectors
			3.2 Creating Stable Transgenic A. thaliana Lines
				3.2.1 Transforming A. thaliana Via Floral  Dip
				3.2.2 Establishing Homozygous Transgenic Lines
			3.3 Characterizing the Transgenic Lines
				3.3.1 Verify Expression of the dCas9-Modifier
				3.3.2 Verify Binding of the dCas9-Modifier to the Locus and its Effect on Chromatin Modifications
				3.3.3 Gene Expression Profiling
		4 Notes
		References
	Chapter 11: Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenom...
		1 Introduction
			1.1 Manipulation of Gene Expression Using Epigenome Editing
		2 Materials
			2.1 CD3+ Cells Isolation and Activation
			2.2 Transduction with CAR-Encoding Retrovirus to Generate CAR T Cells
			2.3 Electroporation of CAR T Cells
			2.4 Assessment of Transduction and Silencing Efficiency as Well as Persistence in CAR T Cells
		3 Methods
			3.1 Generation of CAR T Cells Specific for Prostate Cancer
			3.2 Multiplexed Epigenome Editing in CAR T Cells Via Electroporation
			3.3 Assessment of Transduction and Silencing Efficiency in the Multiplexed Epigenome-Edited CAR T Cells
			3.4 Assessment of Gene Silencing Persistence in the Multiplexed Epigenome-Edited CAR T Cells
		4 Notes
		References
	Chapter 12: Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells
		1 Introduction
			1.1 Establishment of a Fluorescent Reporter System for Chromatin Effector Proteins
			1.2 Application of Reporter Systems to Characterize the Silencing Dynamics of Different Chromatin Effectors
				1.2.1 The Reporter Assay Reflects Chromatin Remodeling
				1.2.2 The Reporter Assay Reflects the Placement of Repressive Modifications
				1.2.3 The Reporter Assay Reflects the Removal of an Activating Histone Modification
				1.2.4 The Reporter System Allows to Analyze Stability and Reversibility of Epigenome Modifications
				1.2.5 The Reporter System Reflects Epigenomic Alterations Induced by the Recruitment of Chromatin Effector Proteins
			1.3 Investigation of Small Molecule Drugs Affecting Chromatin Effector Activity
			1.4 Investigation of the Roles of Essential LSD1 Coregulators Using RNAi
			1.5 Chromatin Effector Coregulator Screen (ChECS) in Reporter Cell Lines to Identify Functional Effector-Coregulator Relations...
			1.6 Summary and Outlook of the Application of Fluorescent Reporter Systems
		2 Materials
			2.1 Cell Culture
			2.2 Transfection
			2.3 Transduction
		3 Methods
			3.1 Maintenance of LentiX Cells
			3.2 Day 1: Transfection of the Packaging Cell Line
			3.3 Day 2: Medium Change
			3.4 Day 3: Transduction of Host Cells
			3.5 Day 4: Medium Change
			3.6 Day 5: Antibiotic Selection and Infection Rate Analysis
			3.7 Day 12: Reporter Cell Line Validation
			3.8 Effector Transduction and Silencing Analysis
		4 Notes
		References
Part IV: Delivery of EpiEditors
	Chapter 13: Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing
		1 Introduction
		2 Materials
			2.1 Cell Culture and Transfection
			2.2 Analysis of Transfected Cells Using Flow Cytometry
		3 Methods
			3.1 Transfection Optimization
			3.2 Harvesting of Cells and Protein Induction Analysis Using Flow Cytometry
		4 Notes
		References
	Chapter 14: Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles E...
		1 Introduction
		2 Materials
			2.1 sgRNA Design
			2.2 Lipid Nanoparticle (LNP) Formulation
			2.3 In Vitro and In Vivo Delivery of mRNA for dCas9 Effectors and gRNA LNP Formulations
			2.4 Visualization of dCas9-Effector Transfection Efficiency In Vitro and In Vivo by Immunofluorescence
		3 Methods
			3.1 gRNA Design
			3.2 dCas9-Effector mRNA Design
			3.3 In Vitro Transcription of dCas9-Effector  mRNA
			3.4 LNP Formulation
				3.4.1 LNP-Mediated In Vitro Delivery of dCas9-Effector mRNA and gRNAs
				3.4.2 Determination of LNP Transfection Efficiency by Immunofluorescence
			3.5 In Vivo Delivery of LNPs Encapsulating RNA for Tumor Targeting
				3.5.1 Inoculation of Tumor Cells
				3.5.2 LNP Encapsulating RNA Preparation for In Vivo Delivery
				3.5.3 Intratumoral/Orthotopic Delivery of LNP Formulations
				3.5.4 Intravenous/Systemic Delivery of LNP Formulations
				3.5.5 Bioluminescence Imaging (BLI) for Control LNPs Incorporating a Luciferase-Encoding  mRNA
				3.5.6 Tumor Measuring and Collection
				3.5.7 Immunofluorescence Staining of Tissue Sections for Detection of dCas9-Effectors in the Target Tissues
		4 Notes
		References
	Chapter 15: Generation of Cell Lines Stably Expressing a dCas9-Fusion or sgRNA to Address Dynamics of Long-Term Effects of Epi...
		1 Introduction
		2 Materials
			2.1 Molecular Biology
			2.2 Plasmids
			2.3 Cell Culture and Transfection
		3 Methods
			3.1 Construction of pHAGE EF1α dCas9-Effector Domain (ED) Vector
			3.2 Transfection and Transduction Protocol to Obtain Stable Expressing dCas9-ED Cell Lines
			3.3 Construction of pMLM2.0-sgRNA Vector
		4 Notes
		References
	Chapter 16: Stereotaxic Surgery as a Method to Deliver Epigenetic Editing Constructs in Rodent Brain
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Instruments and Materials
		3 Methods
			3.1 Neuroepigenetic Editing
			3.2 Validation of Neuroepigenetic Editing Tools
		4 Notes
		References
Part V: Downstream Analysis of Epigenome Editing
	Chapter 17: An Overview of Global, Local, and Base-Resolution Methods for the Detection of 5-Hydroxymethylcytosine in Genomic ...
		1 Introduction
		2 Global Detection of 5hmC Levels
			2.1 Quantifying the Overall Abundance of 5hmC
			2.2 IHC and Flow Cytometry
			2.3 Dot Immunoblotting and ELISA
			2.4 LC-MS/MS
			2.5 Sparse-Coverage Sequencing
		3 Enrichment-Based or Near-Base-Resolution Methods of 5hmC Detection
			3.1 Assessing the Relative or Local Distribution of 5hmC
			3.2 hMeDIP and CMS-DIP
			3.3 GLIB and hmC-Seal
			3.4 Jump-Seq, hmTOP-Seq, and hmC-CATCH
		4 Base-Resolution Localization of 5hmC
			4.1 Mapping 5hmC at the Level of Individual Nucleotides
			4.2 oxBS-Seq
			4.3 TAB-Seq
			4.4 TAPS/TAPS-β and CAPS(+)
			4.5 ACE-Seq and bACE-Seq
			4.6 DIP-CAB-Seq and EBS-Seq
			4.7 Six-Letter  Seq
		5 Emerging Methods of 5hmC Localization
			5.1 The Ever-Expanding Toolbox for Studying 5hmC
			5.2 SMRT Sequencing
			5.3 Nanopore Sequencing
			5.4 Optical Mapping and Single-Molecule Epigenetic Imaging
		6 Conclusions
		References
	Chapter 18: Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation ...
		1 Introduction
			1.1 Summary of the Ultralow Methyl-Seq with TrueMethyl oxBS Protocol
		2 Materials
			2.1 Reagents and Consumables Included in the Ultralow Methyl-Seq Kit with the TrueMethyl oxBS Module (Tecan, Catalog Numbers: ...
			2.2 Additional Equipment, Reagents, and Labware Supplied by the User
			2.3 Data Analysis
		3 Methods
			3.1 DNA Fragmentation
			3.2 DNA Purification
			3.3 End Repair
			3.4 Ligation
			3.5 Final Repair
			3.6 DNA Purification and Denaturation
			3.7 Oxidative Reaction (See Note 17)
			3.8 Bisulfite Conversion (See Note 20)
			3.9 Desulfonation and Purification of Bisulfite-Converted  DNA
			3.10 Determination of the Optimal Amplification Cycles by qPCR (See Note 24)
			3.11 PCR Amplification
			3.12 Purification of the Amplified Library
			3.13 Quantitative and Qualitative Assessment of the Library (See Note 25)
			3.14 Library Normalization and Sequencing
			3.15 Data Analysis
		4 Notes
		References
	Chapter 19: Generation of Whole-Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis
		1 Introduction
		2 Materials
			2.1 Reagents Required for Library Preparation
			2.2 Solutions
		3 Methods
			3.1 Library Preparation
		4 Notes
		References
	Chapter 20: Approaches for the Analysis and Interpretation of Whole-Genome Bisulfite Sequencing Data
		1 Introduction
		2 Materials
			2.1 Software
			2.2 Hardware
			2.3 Data
		3 Methods
			3.1 Preparation
				3.1.1 Install Software
				3.1.2 Trim Reads
				3.1.3 Compress Reads
				3.1.4 Sequencing Quality Control
				3.1.5 Genome Preparation
				3.1.6 Build the Genome Index
			3.2 Alignment
				3.2.1 Align Reads
				3.2.2 Sort BAM  File
				3.2.3 Remove PCR Duplicates
				3.2.4 Remove Intermediate Files
			3.3 Quantifying DNA Methylation
				3.3.1 Call DNA Methylation
			3.4 Post-Alignment Quality Control
				3.4.1 Assess Methylation Bias in Read Position
				3.4.2 Assess Alignment Statistics
				3.4.3 Assess Non-Conversion Rate
				3.4.4 Assess Genome-Wide Methylation Average in Each Context
			3.5 Differential DNA Methylation
				3.5.1 Pre-Processing DNA Methylation Data
				3.5.2 Find Differentially Methylated Positions
				3.5.3 Find Differentially Methylated Regions
			3.6 Interpretation
				3.6.1 Data Visualization
				3.6.2 Interpreting  DMRs
		4 Notes
		References
	Chapter 21: Amplicon-Based Bisulfite Conversion-NGS DNA Methylation Analysis Protocol
		1 Introduction
		2 Materials
			2.1 Isolation of gDNA
			2.2 Bisulfite Conversion of gDNA
			2.3 PCR Optimization/PCR1
			2.4 PCR2
			2.5 Optional PCR Product Purification/Pooling of Samples into the NGS Library
		3 Methods
			3.1 Isolation of gDNA
			3.2 Bisulfite Conversion of gDNA
			3.3 PCR1
			3.4 PCR2
			3.5 (Optional) Purification Before Pooling of Samples for NGS
			3.6 Pooling of the Samples into an NGS Library
			3.7 Data Analysis
		4 Notes
		References
	Chapter 22: Summary of ChIP-Seq Methods and Description of an Optimized ChIP-Seq Protocol
		1 Introduction
			1.1 History of ChIP-Seq
				1.1.1 ChIP-on-Chip with DNA Arrays
				1.1.2 ChIP-qPCR
				1.1.3 ChIP-Seq with SBS
				1.1.4 Other Approaches
			1.2 Recent Developments in ChIP-Seq
				1.2.1 Novel Applications of ChIP-Seq
				1.2.2 ChIP-Seq Data Analysis Algorithms
				1.2.3 Single-Cell ChIP-Seq (scChIP-Seq)
			1.3 Complementary Alternatives to ChIP-Seq
				1.3.1 CUT&RUN
				1.3.2 CUT&Tag
			1.4 Future Directions
		2 Materials
			2.1 Crosslinking of Cells
			2.2 Cell Lysis
				2.2.1 Yeast Cell Lysis
				2.2.2 Human Cell Lysis
			2.3 Chromatin Fragmentation
				2.3.1 MNase Digestion
				2.3.2 Sonication
			2.4 Immunoprecipitate Chromatin and Purify DNA
			2.5 DNA Analysis
				2.5.1 (SYBR GREEN qPCR) (Optional)
				2.5.2 DNA Analysis (Probe-Based qPCR) (Optional)
				2.5.3 DNA Analysis (NGS Sequencing) (Optional)
		3 Methods
			3.1 Protein-DNA Crosslinking
				3.1.1 Yeast Cell Crosslinking
				3.1.2 Human Cell Crosslinking
			3.2 Cell Lysis
				3.2.1 Yeast Cells
				3.2.2 Human Cells
			3.3 Chromatin Fragmentation
				3.3.1 MNase Digestion
				3.3.2 Sonication
			3.4 Preparation of Magnetic Beads and Antibody Mixture
			3.5 Chromatin IP and Purification of DNA
			3.6 DNA Analysis
				3.6.1 ChIP-qPCR (SYBR Green)
				3.6.2 ChIP-qPCR (Probe-Based Assay)
				3.6.3 ChIP-Seq Library Preparation
		4 Notes
		References
	Chapter 23: Single-Molecule Analysis of Transcription Dynamics to Understand the Relationship Between Epigenetic Alterations a...
		1 Introduction
		2 Materials
			2.1 Cell Treatment with Epigenetic and Endocrine Drugs
			2.2 smRNA Fluorescence In Situ Hybridization (FISH)
			2.3 Microscopy Equipment and Imaging Analysis
		3 Methods
			3.1 Cell Culture, Epigenetic, and Endocrine Drug Treatment
			3.2 Probe Design and Handling
			3.3 Fixation and Permeabilization
			3.4 Hybridization with FISH Probes
			3.5 Microscopy Imaging and Image Analysis
		4 Notes
		References
Index