Base Editors: Methods and Protocols

Description

This volume explores base editors (BEs), an invaluable CRISPR-based genome editing tool with a wide variety of versatile applications. Beginning with an overview of BEs, their diverse variants, and computational tools, the book continues with experimental applications of BEs for disease modeling in mammalian cells and generating mutagenic mice, therapeutic base editing strategies, which covers delivery methods of BE-encoded DNA plasmids, mRNAs, or ribonucleoproteins through viruses or non-viral lipid nanoparticles, and lastly, the use of BEs in plants and bacteria. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Base Editors: Methods and Protocols serves as an ideal guide for researchers looking to use base editors to continue their studies in an array of fields. Preface Contents Contributors Part I: Overview of BEs, Diverse Variants of BEs, and Computational Tools for BEs Chapter 1: Introduction and Perspectives of DNA Base Editors 1 Introduction 2 The Construction of Cytosine Base Editors (CBEs) and the Mechanism of Cytosine Base Editing 3 The Construction of Adenine Base Editors (ABEs) and the Mechanism of Adenine Base Editing 4 Improvement of DNA Base Editors 4.1 Expanding Targetable Sites (Table 1) 4.2 Modification of Editing Windows 4.3 Generating Transversion Edits 4.4 Generating Dual Base Conversions of C-to-T and A-to-G at Once 4.5 TC-Specific Base Editing by ABE 4.6 Adeno-Associated Virus (AAV)-Loadable BEs 5 Conclusions References Chapter 2: Web-Based Computational Tools for Base Editors 1 Introduction 2 Materials 2.1 Web Browser 3 Methods 3.1 Guide RNA Design Tool: BE-Designer 3.2 Efficiency Prediction Tool: DeepBaseEditor 3.3 Outcome Prediction Tool: BE-Hive 3.4 Analysis Tool: BE-Analyzer 3.5 Analysis Tool: CRISPResso2 4 Notes References Chapter 3: Prediction of Base Editing Efficiencies and Outcomes Using DeepABE and DeepCBE 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 4: Profiling Genome-Wide Specificity of dCpf1 Cytidine Base Editors Using Digenome-Seq 1 Introduction 2 Materials 2.1 crRNA DNA Templates 2.2 Genomic DNA Extraction 2.3 In Vitro Digestion of Genomic DNA 2.4 Whole-Genome Sequencing 2.5 Digenome-Seq Analysis 3 Methods 3.1 Preparation of the crRNA 3.2 In Vitro Deamination of Genomic DNA 3.3 Digenome-Sequencing to Profile dLbCpf1-BE and USER Mediated Genome-Wide SSBs 3.4 Digenome-Seq Analysis 4 Notes References Part II: Application of BEs for Disease Modeling Chapter 5: Base Editing of Human Hematopoietic Stem Cells 1 Introduction 2 Materials 2.1 Human CD34+ HSPCs 2.2 mRNA Production 2.3 Base Editor Protein 2.4 Lonza 4D Nucleofector 2.5 Polymerase Chain Reaction 3 Methods 3.1 CD34+ Selection 3.2 mRNA Production via In Vitro Transcription 3.3 Protein Expression and Purification 3.4 mRNA Electroporation 3.5 RNP Electroporation 3.5.1 Small-Scale RNP Solution Preparation 3.5.2 Large-Scale RNP Solution Preparation 3.5.3 Electroporation 3.6 Measurement of Base Editing 4 Notes References Chapter 6: A/C Simultaneous Conversion Using the Dual Base Editor in Human Cells 1 Introduction 2 Materials 2.1 Plasmid Preparation 2.2 Cell Transfection 2.3 Sequencing 3 Methods 3.1 Plasmid Preparation 3.1.1 Preparation of LB Medium 3.1.2 Plasmid Construction (Targeting HBG1/2 Site) (See Note 1) 3.2 Cell Transfection 3.2.1 Preparation of 10x PBS Buffer 3.2.2 Transfection Reagent (PEI) Preparation 3.2.3 Transfection 3.3 Preparation of Genomic DNA for Next-Generation Sequencing 3.3.1 Preparation of HI-TOM Mix4 3.3.2 DNA Amplification 3.4 NGS Data Analysis 4 Notes References Chapter 7: Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors 1 Introduction 2 Materials 2.1 DNA Constructs 2.2 Cloning Components 2.3 Transfection and Transduction Components 2.4 Measurement of the Genome Manipulation Efficiency 3 Methods 3.1 BE4max Expressing Cell Line Development (See Note 1) 3.2 BRCA VUS Targeting gRNA Design and Cloning (See Note 2) 3.3 Introduction of BRCA1 Mutation 3.4 NGS Analysis 3.5 Measurement of the Base Substitution Efficiency Using T7 Endonuclease I (T7E1) Assay 3.6 Functional Assessment of BRCA1 Variants 4 Notes References Chapter 8: Use of the Representative Base Editing Tool Target-AID to Introduce Pathogenic Mutations into Mice 1 Introduction 2 Materials 2.1 PCR to Prepare Templates for In Vitro Transcription 2.2 In Vitro Transcription of Target-AID mRNA 2.3 Microinjection to Mouse Zygotes 2.4 Genotyping of Blastocysts and Mice 2.5 Analysis of Off-Target Mutations 3 Methods 3.1 Preparation of Target-AID mRNA 3.2 Preparation of sgRNAs 3.3 Microinjection of Cytoplasm of Mouse Zygotes to Evaluate Efficiency at Blastocyst Stage (See Note 4) 3.4 Genotyping at Blastocyst Stage 3.5 Genotyping of Mice 3.6 Analysis of Off-Target Mutations 4 Notes References Chapter 9: Targeted Mutagenesis in Mice Using a Base Editor 1 Introduction 2 Materials 2.1 Construction of the Single Guide RNA (sgRNA) 2.1.1 Sequences of the Oligos Used for IVT 2.1.2 Template Preparation for IVT 2.1.3 IVT 2.2 Construction of the Base Editor mRNA 2.2.1 Vector Preparation for IVT (with T7 Promoter) 2.2.2 Template Preparation for IVT 2.2.3 IVT 2.3 Mice 2.4 Preparation of the Culture Dishes and Medium 2.5 Induction of Superovulation in Mice 2.6 Collection of 1-Cell Stage Mouse Embryos 2.7 Microinjection 2.8 Vasectomy of Male Mouse 2.9 Embryo Transfer 2.10 Genotyping 2.11 Off-Target Analysis 3 Methods 3.1 Design of the Target sgRNA 3.2 IVT 3.2.1 Production of the Base Editor mRNA via IVT 3.2.2 Production of the sgRNA via IVT 3.3 Collection of 1-Cell Stage Mouse Embryos (See Fig. 2) 3.4 Microinjection 3.4.1 Preparation of the Holding Needle 3.4.2 Preparation of the Injection Needle 3.4.3 Microinjection into the Pronucleus 3.5 Vasectomy of Male Mouse 3.6 Embryo Transfer (See Figs. 2 and 5) 3.7 Genotyping via NGS or Sanger Sequencing (See Fig. 6) 3.7.1 Assessment of the Base Editing Efficiency by Analyzing Preimplantation Blastocyst (Fig. 6) 3.7.2 Genotyping Mice (See Fig. 6) 3.8 Analysis of Off-Target Effects 3.8.1 Off-Target Analysis of DNA 3.8.2 Off-Target Analysis of RNA 4 Notes References Part III: Therapeutic Base Editing Strategies Chapter 10: Heterologous Expression and Purification of a CRISPR-Cas9-Based Adenine Base Editor 1 Introduction 2 Materials 2.1 Protein Expression 2.2 Protein Purification 2.2.1 Lysis and Clarification 2.2.2 Affinity Chromatography (His-Tag Affinity Chromatography) 2.2.3 Intermediate Purification (Flag (M1) Affinity Chromatography) 2.2.4 Final Polishing (Size Exclusion Chromatography) 3 Methods 3.1 Protein Expression 3.2 Protein Purification 3.2.1 Lysis and Clarification 3.2.2 Affinity Chromatography (His-Tag Affinity Chromatography) 3.2.3 Intermediate Purification (Flag (M1) Affinity Chromatography) 3.2.4 Final Polishing (Size Exclusion Chromatography) 4 Notes References Chapter 11: Delivering Base Editors In Vivo by Adeno-Associated Virus Vectors 1 Introduction 2 Materials 2.1 sgRNA Cloning 2.2 Evaluating Base Editing in Mammalian Cells 2.3 AAV Vector Packaging 2.4 AAV Vector Titering 2.5 Intrathecally Delivering AAV Vector to Mice for In Vivo DNA Editing 2.6 Measuring Base Editor Expression and DNA Editing 3 Methods 3.1 Constructing a Base Editing System 3.1.1 Designing and Cloning the sgRNA 3.2 Evaluating Base Editing in Mammalian Cells 3.2.1 Transfection 3.2.2 Analyzing Base Editing Outcomes 3.3 AAV Packaging 3.3.1 Transfection 3.3.2 Harvesting 3.3.3 Iodixanol Gradient Purification 3.3.4 AAV Buffer Exchange and Concentration 3.3.5 Titering by qPCR 3.4 Intrathecally Delivering AAV Vector to Mice for In Vivo DNA Editing 3.4.1 Intrathecal Injection 3.4.2 Tissue Collection 3.5 Measuring Base Editor Expression and DNA Editing 3.5.1 Measuring Base Editor Expression by Immunohistochemistry 3.5.2 Verification of In Vivo Base Editing by NGS 4 Notes References Chapter 12: The Delivery of ABE mRNA to the Adult Murine Liver by Lipid Nanoparticles (LNPs) 1 Introduction 2 Materials 2.1 Plasmids for DNA Template 2.2 Restriction Enzymes and a Reaction Buffer 2.3 Gel Analysis 2.4 Purification for Nucleic Acid 2.5 Transcription Enzymes and Reagents 2.6 Chemical Materials and Instruments for LNPs Fabrication 2.7 Mice, Reagent, and Supplies for In Vivo Delivery 3 Methods 3.1 DNA Template Preparation 3.1.1 Prepare ABE6.3 Template 3.1.2 Prepare gRNA Template 3.2 In Vitro Transcription 3.2.1 ABE6.3 mRNA In Vitro Transcription 3.2.2 Poly(A) Tailing of ABE6.3 mRNA 3.2.3 In Vitro Transcription and Purification of gRNAs 3.3 Lipid Nanoparticles (LNPs) Package 3.3.1 Microfluidic Device Fabrication 3.3.2 Microfluidic Device Test 3.3.3 Synthesis of LNPs 3.3.4 Characterization of LNPs 3.4 Delivery to the Murine Liver 4 Notes References Chapter 13: Ex Vivo Base Editing Therapy with Chemically Derived Hepatic Progenitors 1 Introduction 2 Materials 2.1 Primary Hepatocyte Isolation Procedure from Genetic Disorder Model Mice 2.2 Reprogramming and Maintenance Procedure 2.3 Base Editing and Selection Procedure 2.4 Transplantation 3 Methods 3.1 Primary Hepatocyte Isolation from Mouse Liver Tissue 3.2 Generation and Maintenance of Chemically Derived Hepatic Progenitors 3.3 Base Editing and Selection of Chemically Derived Hepatic Progenitors 3.4 Transplantation of Cloned CdHs into Mice 4 Notes References Chapter 14: Application of Base Editor-Mediated Genome Editing in Mouse Retina 1 Introduction 2 Materials 2.1 Viral Vector Production 2.2 Base Editor Ribonucleoprotein (RNP) Construction 2.3 Equipment 2.4 Sterile Plastic and Glassware 2.5 Reagent 2.6 Experimental Animals 3 Methods 3.1 Preparation of Viral Vector 3.2 Preparation of RNP/Lipofectamine Mixture 3.3 Preparation of Injection Kit 3.4 Injection 3.5 Sample Preparation 3.6 Immunofluorescence (Optional, See Note 10) 3.7 Evaluation of Editing Efficiency 4 Notes References Part IV: Use of BEs in Plants and Bacteria Chapter 15: High-Throughput Base Editing-Mediated Artificial Evolution Streamlines Trait Gene Identification in Rice 1 Introduction 2 Materials 2.1 Prepare Immature Seeds 2.2 Vector Preparation 2.3 Rice Transformation 2.4 Preparing for Identification of Mutation and Phenotype 3 Methods 3.1 Select the Appropriate Base Editor 3.2 Design Vectors 3.3 Agrobacterium Infection 3.4 Plant Regenerate 3.5 Screen and Genotype 4 Notes References Chapter 16: Heritable Virus-Induced Genome Editing (VIGE) in Nicotiana attenuata 1 Introduction 2 Materials 2.1 Generation of SpCas9-Overexpressed N. attenuata 2.2 Preparation of Guide RNA Delivery Vector 2.3 Agroinfiltration 2.4 Targeted Deep Sequencing 2.5 Instruments 3 Methods 3.1 Generation of SpCas9-Overexpressed N. attenuata 3.2 Guide RNA (gRNA) Design 3.3 TRV2-PEBV-gRNA Cloning 3.4 Agroinfiltration 3.5 Design PCR Primers for Targeted Deep Sequencing 3.6 Validation of Editing Efficiency in Infiltrated Leaves 3.7 Validation of Editing Efficiency in Progeny 4 Notes References Chapter 17: Cytosine Base Editing in Bacteria 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Media 2.3.1 Media Used for C. glutamicum Cultivation 2.3.2 Media Used for B. subtilis Cultivation 2.4 Reagents and Kits 2.5 Primers 3 Methods 3.1 gRNA Design 3.2 Plasmid Construction for Base Editing 3.3 Strain Transformation 3.3.1 Transformation of C. glutamicum with the Constructed Base Editing Plasmid 3.3.2 Transformation of B. subtilis 168 with the Constructed Base Editing Plasmid 3.4 Verification and Isolation of Base-Edited Strains 3.5 Plasmid Curing 4 Notes References Index