Plant Genome Engineering: Methods and Protocols

Preface
Contents
Contributors
Part I: Genome Engineering Systems
Chapter 1: PAM-Less CRISPR-SpRY Genome Editing in Plants
1 Introduction
2 Materials
3 Methods
3.1 T-DNA Vector Construction for CRISPR-SpRY-Mediated Genome Editing
3.2 Rice Protoplast Transformation to Test the Editing Efficiency of SpRY Vectors
4 Notes
References
Chapter 2: Type I-D CRISPR System-Mediated Genome Editing in Plants
1 Introduction
2 Materials
2.1 Design of crRNA Targeting a Specific Sequence
2.2 Construction of a crRNA Vector for Expression in Mammalian Cells
2.3 HEK293T Cell Culture
2.4 Transfection of HEK293T Cells for NanoLuc SSA Reporter Assay
2.5 Detection of Luciferase Activity
2.6 Construction of a Plant-Optimized TiD Vector Containing a crRNA Targeting a Specific Sequence
2.7 Germination of Tomato Seeds
2.8 Transformation of Agrobacterium tumefaciens Using the TiD Vector
2.9 Preparation of Agrobacterium Solution
2.10 Agrobacterium-Mediated Transformation of the TiD Vector into Tomato Leaf Discs
2.11 Regeneration and Cultivation of Transgenic Tomatoes
3 Methods
3.1 Design crRNA Targeting a Specific Sequence
3.2 Construction of crRNA Expression Vector for Mammalian Cells
3.3 HEK293T Cell Culture
3.4 Transfection of HEK293T Cells for NanoLuc SSA Reporter Assay
3.5 Detection of Luciferase Activity (See Note 13)
3.6 Construction of a Plant-Optimized TiD Vector Containing a crRNA that Targets the Specific Sequence
3.7 Germination of Tomato Seeds
3.8 Transformation of A. tumefaciens Using TiD Vector
3.9 Preparation of Agrobacterium Solution
3.10 Agrobacterium-Mediated Transformation of Tomato Leaf Discs with TiD Vector
3.11 Regeneration and Cultivation of Transgenic Tomatoes (Fig. 5a)
4 Notes
References
Chapter 3: CRISPR/LbCas12a-Mediated Genome Editing in Soybean
1 Introduction
2 Materials
2.1 Laboratory Supplies
2.2 Equipment
2.3 Chemicals, Media Recipes, and Reagents
2.3.1 Chemicals
2.3.2 Media
2.3.3 Other Supplies
2.3.4 Disarmed Agrobacterium tumefaciens Strains
2.3.5 Transformation and Genome Editing Vectors
2.3.6 Plant Materials
3 Methods
3.1 Seed Sterilization
3.2 Seed Germination
3.3 Agrobacterium Preparation
3.4 Explant Preparation
3.5 Explant Infection
3.6 Cocultivation
3.7 Shoot Induction and Selection
3.8 Shoot Elongation
3.9 Transplanting and Rooting
3.10 Transgene Copy Number and Target Editing Analysis
3.11 T0 Plant Growth and Seed Production
3.12 Examples of Experiment Results
4 Notes
References
Chapter 4: Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Equipment
2.3 Regents
2.4 Solutions and Media
3 Methods
3.1 Editing Targets and gRNA Design
3.2 T-DNA Vector Construction for CBE and ABE Systems
3.3 Agrobacterium-Mediated Poplar Transformation
3.4 Analysis of Base Editing Efficiency
4 Notes
References
Chapter 5: Genetic Engineering of Potato (Solanum tuberosum) Chloroplasts Using the Small Synthetic Plastome ``Mini-Synplastom...
1 Introduction
2 Materials
2.1 Supplies and Solutions for Gold Particle Preparation
2.1.1 Supplies
2.1.2 Solutions and DNA
2.2 Media and Solutions for Potato Tissue Culture
2.2.1 Stock Solutions and Supplies
2.2.2 Media for Regeneration Synplastomic Plants and In Vitro Propagation
2.3 Materials and Methods for Genotyping
2.4 Equipment
3 Methods
3.1 Design the Mini-synplastome for Chloroplast Engineering in Potato
3.2 Gold Particle Preparation
3.3 Biolistic Transformation of Potato Leaves and Regeneration of Synplastomic Plants in Tissue Culture
3.4 Example of Genotyping Synplastomic Plants
4 Notes
References
Part II: Design and Validation Tools
Chapter 6: Designing Guide-RNA for Generating Premature Stop Codons for Gene Knockout Using CRISPR-BETS
1 Introduction
2 Materials
2.1 Dependencies and Software Installation
2.1.1 Install via Precompiled File (Recommended)
2.1.2 Install via Source Code
2.2 Prepare Input File
2.2.1 GenBank
2.2.2 SnapGene
2.2.3 FASTA
3 Methods
3.1 Select and Upload File
3.2 Select the Edit System Option
3.3 Scanning Result Panel and Download Result Information
3.4 Pick the Appropriate gRNA
4 Notes
References
Chapter 7: Construction of CRISPR/Cas9 Multiplex Genome Editing System in Rice
1 Introduction
2 Materials
3 Methods
3.1 Target Sequence Selection and Primer Design
3.2 Construction of Single Target Editing Vector
3.3 Construction of Multi-Target Editing Vector
3.3.1 Construction of Single Intermediate Vector (Fig. 3)
3.3.2 Assemble Three Intermediate Vectors to Binary Vector in One Step (Fig. 4)
3.3.3 Five Intermediate Vectors Assembled to Binary Vector (Fig. 5)
3.4 Genetic Transformation
3.5 Mutation Detection
4 Notes
References
Chapter 8: Use of Fluorescent Protein Reporters for Assessing and Detecting Genome Editing Reagents and Transgene Expression i...
1 Introduction
2 Materials
2.1 Protoplast Transformation
2.2 Leaf Infiltration
2.3 Stable Transformation
3 Methods
3.1 Protoplast Transformation
3.2 Leaf Infiltration
3.3 Stable Transformation
4 Notes
References
Chapter 9: Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposable Consumables
2.3 Plant Materials, Enzymes, and Plasmids
2.4 Chemicals and Stock Solutions
2.5 Buffer and Enzyme Solutions
3 Methods
3.1 Etiolated Maize Seedling Material Preparation
3.2 Isolation of Etiolated Maize Leaf Protoplasts
3.3 Liquid Handler Automated Transfection of Protoplasts
3.4 Genome Editing Detection Assay
3.5 ImageJ Quantification of Editing Efficiency
3.6 Fluorescence Analysis
3.7 Transfection of Soybean Immature Cotyledon Protoplasts
4 Notes
References
Part III: Transgene-Free Delivery of Machinery
Chapter 10: Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line
1 Introduction
2 Materials
3 Methods
3.1 Preparation of LbCas12a Nuclease
3.1.1 LbCas12a Expression Vector Construction
3.1.2 LbCas12a Protein Expression and Isolation
3.2 Preparation of crRNA
3.3 LbCas12a-RNP-Mediated Genome Editing in Transfected Citrus Protoplasts
3.3.1 Citrus Protoplast Isolation and RNP Transfection
3.3.2 Analysis of LbCas12a RNP-Mediated Genome Editing in Citrus Protoplasts
4 Notes
References
Chapter 11: Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector
1 Introduction
2 Materials
2.1 Viral Vectors, Bacterial Strains, and Plant Materials
2.2 Molecular Cloning
2.3 Agroinoculation
2.4 Mutation Identification and Genotyping
2.5 Tissue Culture and Plant Regeneration
2.6 Equipment
3 Methods
3.1 Construction of SYNV-Based CRISPR/Cas9 Vector
3.2 Agroinfiltration with SYNV Vectors (Agroinoculation)
3.3 Analysis of Mutation Frequency
3.4 Plant Regeneration and Genotyping
4 Notes
References
Chapter 12: Ribonucleoprotein (RNP)-Mediated Targeted Mutagenesis in Barley (Hordeum vulgare L.)
1 Introduction
2 Materials
2.1 gRNAs and Cas Enzymes
2.2 Plant Material
2.3 Stock Solutions (See Note 1)
2.3.1 Mineral Salts
2.3.2 Carbohydrate Source
2.3.3 Vitamins
2.3.4 Growth Regulators
2.3.5 Amino Acids
2.3.6 Selective Agents
2.3.7 Gelling Agent
2.3.8 Other Additives and Solutions
2.4 Plant Tissue Culture Media
2.5 Laboratory Supplies
2.5.1 Immature Embryo Isolation
2.5.2 Bombardment of Immature Barley Embryos
2.6 Plant Genomic DNA Isolation
2.7 Adaptor Polymerase Chain Reaction
2.8 Amplicon Sequencing
3 Methods
3.1 Growing the Donor Material for Stable RNP-Mediated Knockouts
3.2 Isolation of Immature Embryos (Day 1)
3.3 RNP Assembly
3.4 Coating of Gold Particles with DNA and RNP Complexes
3.5 Bombardment of Immature Embryos
3.6 Callus Formation, Regeneration, and Mutant Detection (from Day 2 up to 8-12 Weeks)
4 Notes
References
Chapter 13: Ribonucleoprotein (RNP)-Mediated Allele Replacement in Barley (Hordeum vulgare L.) Leaves
1 Introduction
2 Materials
2.1 gRNA, Cas Enzyme, and Repair Template
2.2 Plant Material
2.3 Stock Solutions (See Note 1)
2.3.1 Selective Agents
2.3.2 Gelling Agent
2.3.3 Other Additives and Solutions
2.4 Media for Leaf Bombardment
2.5 Laboratory Supplies
3 Methods
3.1 Growing the Donor Material for Leaf Bombardment
3.2 RNP Assembly
3.3 Coating of Gold Particles with DNA and RNP Complexes
3.4 Bombardment of Barley Leaves
4 Notes
References
Chapter 14: Genome Editing in Chlamydomonas reinhardtii Using Cas9-gRNA Ribonucleoprotein Complex: A Step-by-Step Guide
1 Introduction
2 Materials
3 Methods
3.1 Bioinformatic Analysis
3.1.1 Gene Selection and Sequence Retrieval
3.1.2 Designing of gRNAs
3.1.3 Off-Target Predictions
3.2 Preparation of CRISPR Tools
3.2.1 gRNA Expression Cassette Construction and In Vitro Transcription
3.2.2 Purification of gRNAs
3.2.3 Quantification of gRNAs
3.2.4 Cas-Associated Protein 9 (Cas9) Nuclease
3.2.5 In Vitro Cleavage Demonstration
3.3 Cell Preparation
3.3.1 Culture Scale-Up
3.3.2 Competent Cell Preparation
3.4 Transformation of CRISPR Tools
3.4.1 Formation of Cas9-gRNA RNP Complex
3.4.2 Transformation of CRISPR Tools
3.4.3 Incubation and Growth
3.5 Screening and Sequencing of Clones
3.5.1 Genomic DNA Extraction
3.5.2 Target Fragment Amplification and Cleanup
3.5.3 Sanger Sequencing, Analysis of Sequenced Reads, and Identification of Mutations
4 Notes
References
Part IV: Generation and Analysis of Engineered Lines
Chapter 15: Highly Efficient Gene Knockout in Medicago truncatula Genotype R108 Using CRISPR-Cas9 System and an Optimized Agro...
1 Introduction
2 Materials
2.1 Adding Guides to Vector and Transformation of Agrobacterium tumefaciens
2.2 Medicago truncatula Growth to Produce Explant Material for Agrobacterium Transformation
2.3 Medicago Transformation, T0 Plant Production, and T1 Seed Production
2.3.1 Stock Solutions
2.3.2 Media
2.3.3 Equipment
2.4 Genotyping Transgenic Plants
3 Methods
3.1 Cloning of Guide Sequences into Vector and Transfer into Agrobacterium
3.2 Medicago truncatula Growth to Produce Explant Material for Agrobacterium Transformation
3.3 Medicago Transformation, T0 Plant Production, and T1 Seed Production
3.4 Genotyping T0 Primary Transgenic Plants
3.5 Identification of Transgene-Free Mutant Lines
4 Notes
References
Chapter 16: Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems
1 Introduction
2 Materials
2.1 Cloning
2.2 Genotyping Transgenic Plants
3 Methods
3.1 Selection of Target Sequences
3.2 Cloning SpCas9 Guides
3.3 Cloning of LbCas12a Guides
3.4 Transformation of Guide Digligs into E. coli
3.5 Screening Cloned Guides
3.6 Level 2 Cloning to Make SpCas9 Constructs Containing Between One and Four Guides
3.7 Level 2 Cloning to Make LbCas12a Constructs Containing Between One and Four Guides
3.8 Transformation of Level 2 Digligs into E. coli
3.9 Screening Level 2 Colonies
3.10 Genotyping T0 Primary Transgenic Plants
3.11 Identification of Transgene-Free Mutant Lines
4 Notes
References
Chapter 17: Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr...
1 Introduction
2 Materials
2.1 Bacterial Strains and Plasmid Vectors
2.2 Reagents and Supplies
2.3 Equipment
2.4 Bacterial and Plant Culture Media
3 Methods
3.1 Vector Construction
3.2 GoldenBraid Assembly of Binary Vector
3.3 Plant Transformation and Tissue Culture
3.3.1 Soybean Seed Sterilization and Germination
3.3.2 Agrobacterium Inoculum Preparation
3.3.3 Explant Preparation and Inoculation
3.3.4 Shoot Initiation
3.3.5 Shoot Elongation Step
3.3.6 Rooting Step
3.4 Greenhouse Soybean Growth and Preliminary Phenotyping Assessment
3.5 Genotyping of Primary Events
3.5.1 Genomic DNA Extraction
3.5.2 Genotype Primary Event for Edits
4 Notes
References
Chapter 18: A CRISPR/Cas9 Protocol for Target Gene Editing in Barley
1 Introduction
2 Materials
2.1 Plant Material
2.2 Reagents
3 Procedure
3.1 Donor Plant Growth and Target Gene Cloning (Step 1)
3.2 The SgRNA Design and the Genome Editing Vector Construction (Step 2)
3.3 Agrobacterium-Mediated Genetic Transformation of Barley (Step 3)
3.4 Screening of Transgenic Plants (Step 4)
3.5 Screening of Mutant Plants and Genotypic Analyses (Step 5)
3.6 Off-Target Mutation Analysis (Step 6)
3.7 Genotypic Analyses of Mutant Progeny Plants (Step 7)
3.8 The Selection of the Transgene-Free Mutant Lines (Step 8)
3.9 The Phenotypic Analysis (Step 9)
4 Notes
References
Chapter 19: Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration
1 Introduction
2 Materials
2.1 Supplies and Equipment
2.2 Plant Material and Selection of the Target Site
2.3 Chemicals and Stock Solutions
2.4 Working Solution and Growth Medium
3 Methods
3.1 Explant Preparation
3.2 sgRNA Template Assembly, Amplification, In Vitro Transcription, and sgRNA Purification
3.3 Cas9/sgRNA Ribonucleoprotein Assembly
3.4 Donor DNA Preparation
3.5 Protoplast Isolation
3.6 Protoplast Transfection (Nucleofection)
3.7 Protoplast Regeneration
3.8 Evaluation of CRISPR/RNP Complex-Targeted Insertion Efficiency in Protoplasts and Regenerants
3.8.1 Testing the Initial Protoplast Transfection Efficiency
3.8.2 Genotyping of Regenerated Plants
3.9 Whole-Genome Sequencing for Off-Target Donor DNA Insertions
4 Notes
References
Chapter 20: Stepwise Optimization of Real-Time RT-PCR Analysis
1 Introduction
2 Materials
2.1 Reagents and Supplies
2.2 Equipment
3 Methods
3.1 RNA Isolation and cDNA Synthesis
3.2 Selection of Candidate Reference Genes Based on Their Digital Expression Profiles
3.3 Identification of the Homologous Sequences of Each Candidate Reference Gene in the Genome
3.4 Validation of the Accuracy of the Full-Length Transcript Sequences by PCR and Sanger Sequencing Without Cloning
3.5 Primer Design for qPCR
3.6 Optimization of qPCR Conditions
3.7 qPCR
3.8 Data Analysis for Transcript Abundance of the Candidate Reference Genes
3.9 Analysis of the Stability of Expression of the Candidate Reference Genes
3.10 Identification of the Optimal Number of Reference Genes
4 Notes
References
Chapter 21: CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding
1 Introduction
2 Materials
2.1 General Molecular Biology Supplies
2.2 Bacteria Strains
2.3 Golden Gate Binary Vector Assembly
2.4 Ribonucleoprotein (RNP) Complex Assembly
2.5 Antibiotics and Phytohormone Stock Solutions
2.6 Culture Media
2.7 Molecular Characterization
2.7.1 Target Gene Sequencing
2.7.2 Primers for T-DNA Detection
2.7.3 CAPS
2.7.4 High-Resolution Fragment Analysis (HRFA)
2.8 Other Equipment and Supplies
3 Methods
3.1 Target Gene Sequencing
3.2 Target Site Selection
3.3 Off-Target Site Prediction
3.4 CRISPR/Cas9 Binary Vector Assembly
3.4.1 sgRNA Amplification
3.4.2 Cloning Step 1
3.4.3 Cloning Step 2
3.5 RNP Complex Assembly
3.6 CRISPR/Cas9 System Delivery in Potato
3.6.1 Introduction of the Binary Vector into Agrobacterium tumefaciens
3.6.2 Preparation of Plant Material
3.6.3 Agrobacterium Culture
3.6.4 Explant Infection and Plant Regeneration
3.7 Molecular Analysis of Regenerated Lines
3.7.1 Confirmation of T-DNA Integration into the Potato Genome
3.7.2 CAPS Assay
3.7.3 HRFA
4 Notes
References
Part V: Genome Engineering for Crop Improvement
Chapter 22: Recent Advances in Engineering of In Vivo Haploid Induction Systems
1 Introduction
2 Maternal Haploid Inducers in Corn
3 The Journey to Develop Maternal Haploid Induction System
3.1 Maternal Haploid Genes
3.2 Genome Editing of Haploid Inducer Orthologs in Other Crops
4 The Journey to Develop Paternal Haploid Induction System
4.1 Paternal Haploid Genes
4.2 Paternal Haploid Inducer Development via Genome Editing
5 The Needs for Genome Editing Tools
6 Perspectives
References
Index