RNA-Based Technologies for Functional Genomics in Plants (Concepts and Strategies in Plant Sciences)

Preface
Contents
Editors and Contributors
	About the Editors
	Contributors
1 Artificial Small RNAs for Functional Genomics in Plants
	1.1 Introduction
	1.2 Artificial sRNAs (Art-sRNAs)
		1.2.1 Artificial microRNAs (amiRNAs)
		1.2.2 Artificial/Synthetic Trans-Acting Small Interfering RNAs (atasi/syn-tasiRNAs)
	1.3 Design, Production, and Validation of Art-sRNA Constructs
		1.3.1 Design of Plant Art-sRNAs
		1.3.2 Generation of Art-sRNA Constructs
		1.3.3 In Vivo Validation of Art-sRNA Constructs
	1.4 Examples of Art-SRNAs Used in Gene Function Studies in Plants
		1.4.1 Artificial MiRNAs
		1.4.2 Artificial/Synthetic tasiRNAs
	1.5 Concluding Remarks and Future Perspectives
	References
2 Recent Advancements in MIGS Toward Gene Silencing Studies in Plants
	2.1 Introduction
	2.2 Biogenesis of MicroRNA-Triggered Secondary siRNAs
	2.3 Gene Silencing Technologies Based on tasiRNA Pathway
	2.4 MicroRNA-Induced Gene Silencing (MIGS) and Its Advantages
	2.5 Gene Silencing Studies in Plants Using MIGS
	2.6 Limitations of MIGS and Steps to Overcome the Limitations
	2.7 Conclusions
	References
3 Target Mimic and Short Tandem Target Mimic Technologies for Deciphering Functions of miRNAs in Plants
	3.1 Introduction
	3.2 TM and STTM
	3.3 Construction of STTM
		3.3.1 Design of STTM Structure
		3.3.2 Promoter Selection
		3.3.3 Plasmid Construction
	3.4 Application of STTM
	References
4 Silencing and Expressing MicroRNAs in Plants Through Virus-Based Vectors
	4.1 Introduction
	4.2 Strategies for Functional Analysis of MicroRNAs
	4.3 Virus-Based Gene Silencing or Gene Overexpression in Plant
	4.4 Virus-Based miRNA Expression or Silencing in Plants
	4.5 Conclusions and Future Perspectives
	References
5 Use of mRNA-Interactome Capture for Generating Novel Insights into Plant RNA Biology
	5.1 Introduction: RNA-Binding Proteins Execute Post-Transcriptional Regulation
	5.2 RBPs Are an Understudied Class of Gene Regulators
	5.3 The Global Identification of RBPs with mRNA-Interactome Capture
	5.4 Arabidopsis in Planta mRNA-Interactome Capture
	5.5 The Use of mRNA-Interactome Capture to Address Key Areas of Plant Biology
	5.6 Conclusions
	References
6 Slicing Messengers by Artificial Designs: Artificial MicroRNA Induced Gene Silencing in Polyploid Plants for Functional Genomics and Trait Modification
	6.1 Introduction
		6.1.1 Polyploidy, Gene Redundancy, and Challenge of Functional Characterization
		6.1.2 Principles of Natural Small RNAs and Gene Silencing Phenomenon
	6.2 Artificial microRNA Based Tools: Design and Engineering
		6.2.1 Web MicroRNA Designer
		6.2.2 The Plant Small RNA Maker Suite (P-SAMS)
		6.2.3 amiRNA Design/MicroRNA Designer
		6.2.4 amiRNA Designer
	6.3 Artificial miRNA-Mediated Gene Silencing: Case Studies on Functional Genomics and Trait Modification
		6.3.1 Gene Silencing in Polyploid System: Brassica Species as Case Study
		6.3.2 Functional Genomics
	6.4 Advances in AmiRNA Technology
	6.5 Summary and Conclusions
	References
7 Suppressor to Survival: RNAi as a Molecular Weapon in Arms Race Between Virus and Host
	7.1 Introduction
	7.2 Plant Antiviral Defense Mechanism
		7.2.1 PTGS as Antiviral Defense
		7.2.2 TGS as Antiviral Defense
	7.3 Small RNA Pathways in Antiviral Defense
		7.3.1 Virus Induced Gene Silencing
		7.3.2 MicroRNA as an Antiviral Defense
		7.3.3 Suppressor of RNAi
	7.4 Approaches for the Identification of RSS
		7.4.1 Agroinfiltration and Reversal Assays
		7.4.2 Reversal of Transgene Induced Silencing
		7.4.3 Crossing Assay
		7.4.4 Grafting Assay
	7.5 Mechanism of RNA Silencing Suppression
		7.5.1 Binding of Long dsRNAs: Inhibition of the Dicing Steps
		7.5.2 Binding to Biogenesis Components
		7.5.3 Viral Suppressors Preventing RISC Assembly
		7.5.4 Interference with DNA Methylation
	7.6 Components of RNAi Silencing Machinery
		7.6.1 Cis-Acting siRNAs as a Silencing Tool
		7.6.2 Trans-Acting siRNAs as a Silencing Tool
		7.6.3 Natural cis-Acting siRNAs as a Silencing Tool
		7.6.4 Anti-sense RNA
		7.6.5 Targeting RNA Components of Silencing
		7.6.6 Targeting Protein Components of Silencing
		7.6.7 Modifying Expression of Host Genes
	7.7 Implications for RNA Silencing Suppressor
		7.7.1 RSSs as Tools Unraveling the Molecular Basis of Silencing
		7.7.2 RSSs as Molecular Probes
		7.7.3 Enhancing Transgene Expression
		7.7.4 Development of Antiviral Strategies
		7.7.5 Molecular Farming
	7.8 Conclusions
	References
8 An Improved Virus-Induced Gene Silencing (VIGS) System in Zoysiagrass
	8.1 Introduction
	8.2 Materials
		8.2.1 Amplification of Target Gene
		8.2.2 Cloning Target Gene into Vector
		8.2.3 Agrobacterium Transformation
		8.2.4 Inoculation
	8.3 Methods
		8.3.1 Preparation of Plant Materials
		8.3.2 Amplification of Target Gene Fragment (TGF)
		8.3.3 Cloning into TRV2-LIC Vector
		8.3.4 Preparation of Agrobacterium Used in VIGS
		8.3.5 Agro-Infiltration of N. Benthamiana
		8.3.6 Inoculation of Zoysiagrass
		8.3.7 Evaluation of Gene Silencing
	References
9 RNA Interference (RNAi): A Genetic Tool to Manipulate Plant Secondary Metabolite Pathways
	9.1 Introduction
	9.2 Metabolic Engineering
	9.3 RNA Interference (RNAi)
		9.3.1 Mechanism
		9.3.2 Vector and Transformation Methods
	9.4 Conclusion
	References
10 Improving Nutrient Value of Crops: Applications of RNAi in Targeting Plant Metabolic Pathways
	10.1 Introduction
	10.2 Crop Improvement
	10.3 Metabolic Engineering
		10.3.1 Generation of End Product of Metabolic Pathway
		10.3.2 Accumulating an Intermediate Product
		10.3.3 Strategies to Alleviate Several Compounds Simultaneously
	10.4 Tools for Metabolic Engineering and RNA-Based Technologies as a Promising Approach
		10.4.1 Development of Customized Systems for Overproduction of Plant Products
		10.4.2 Engineering New Traits into Crops
		10.4.3 Genetic Approaches
		10.4.4 RNA-Based Technologies as an Emerging Approach
	10.5 Mechanism of RNAi
		10.5.1 Generalized Strategy of RNA Interference
		10.5.2 Pathways Operating in Plants for RNA Silencing
		10.5.3 Transformation Methods for RNAi Constructs into Plants
	10.6 Applications of RNAi
		10.6.1 Biofortification
		10.6.2 High Amylose Starch Production by RNAi
		10.6.3 RNAi in Oilseed Improvement
		10.6.4 RNAi in Hypoallergenic Plant
		10.6.5 Reduction of Alkaloid in Crops by RNAi
	10.7 Conclusions
	References
11 Gene and Genome Editing with CRISPR/Cas Systems for Fruit and Vegetable Improvement
	11.1 From Chromosome Transfer to Single Gene Transfer
	11.2 Gene Targeting
	11.3 First-Generation Genome Editing Technologies
	11.4 Zinc Finger Nuclease Genome Editing
	11.5 TALEN-Based Genome Editing
	11.6 The CRISPR/Cas Technologies
	11.7 Types of CRISPR/Cas Systems
		11.7.1 CRISPR/Cas9
		11.7.2 Crispr/CAS12a (Cpf1)
		11.7.3 Crispr/Cas 13(C2c2)
		11.7.4 Using Crispr to Modify Single Genes
	11.8 Using Crispr to Modify Protein Families and Complex Genomes
	11.9 Conclusions
	References
12 Principles and Applications of RNA-Based Genome Editing for Crop Improvement
	12.1 Introduction
		12.1.1 Genome Editing—As Molecular Scissors of Mutation
		12.1.2 Tools for Genome Editing
	12.2 CRISPR/Cas System—A Wide Horizon of Genome Editing
		12.2.1 CRISPR/Cas9
		12.2.2 CRISPR/Cas12a (Cpf1)
		12.2.3 CRISPR/Cas13
		12.2.4 Base Editing
		12.2.5 Multiplexing
		12.2.6 CRISPR—Off-Targets
		12.2.7 Delivery Methods of CRISPR Cassette
		12.2.8 Engineered Cas9 Modifications
	12.3 CRISPR—For Revamping Plant Growth and Development
		12.3.1 Yield and Grain Quality Enhancement
		12.3.2 Tackling Abiotic Stresses
		12.3.3 Defending Against Biotic Stressors
		12.3.4 Other Key Applications of CRISPR
	12.4 Regulatory Aspects on CRISPR Plants
	12.5 Conclusion with Perspectives
	References
13 CRISPR-Cas12a (Cpf1) and Its Role in Plant Genome Editing
	13.1 Introduction
	13.2 Where Does CRISPR Come from
	13.3 Discovery and Characterization of the CRISPR-Cas12a System
	13.4 Other CRISPR Systems
	13.5 How CRISPR Functions
	13.6 Comparison of CRISPR-Cas9 and CRISPR-Cas12a Systems
	13.7 Applications of Cas12a (Cpf1) in Plant Genome Editing
		13.7.1 Cotton
		13.7.2 Maize (Corn) and Sorghum
		13.7.3 Rice
	13.8 Looking Forward
	References
14 CRISPR/Cas13: A Novel and Emerging Tool for RNA Editing in Plants
	14.1 Introduction
	14.2 CRISPR/Cas System
		14.2.1 Discovery and Mechanism
		14.2.2 Applications of CRISPR/Cas System
		14.2.3 Classification of CRISPR/Cas System
	14.3 CRISPR/Cas Type VI System (Cas13)
		14.3.1 Discovery
		14.3.2 Evolutionary Scenario for Type VI CRISPR/Cas Systems
		14.3.3 Variations of CRISPR/Cas Type VI System (Cas13)
		14.3.4 Mechanism of Type VI CRISPR/Cas System
		14.3.5 Potential Applications of CRISPR/Cas13
	14.4 Potential Limitations of CRISPR/Cas13
	14.5 Future Prospects
	References
15 Mutagenomics for Functional Analysis of Plant Genome using CRISPR Library Screen
	15.1 Introduction
	15.2 CRISPR/Cas9-Mediated Targeted Mutagenesis in Plants
	15.3 Construction of the CRISPR Library
	15.4 Screening Methods
		15.4.1 Pooled CRISPR Screening
		15.4.2 Arrayed CRISPR Screening
		15.4.3 High-Resolution Fragment Analysis (HRFA) for Screening of Mutations
		15.4.4 Mutation Sites-Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR)
		15.4.5 CRISPR Interference (CRISRi) Screening
		15.4.6 CRISPR Activation (CRISPRa) Screening
		15.4.7 Dual Screening
		15.4.8 ResponderSCREEN
		15.4.9 CRISPR/Cas9 Target Essentiality Screening (CTEs)
	15.5 Generation of Transgenic Plants Using CRISPR Library
	15.6 DNA-Free Genome Editing
	15.7 Conclusion and Future Implications
	References
16 CRISPR/Cas9 System, an Efficient Approach to Genome Editing of Plants for Crop Improvement
	16.1 Introduction
	16.2 CRISPR/Cas System; Origin, Mechanism, and Its Use in Genome Editing
	16.3 Effectiveness and Versatility of CRISPR/Cas9 System in Genome Editing of Plants
	16.4 Genome Editing Technologies Contribute to Pathogen Resistance in Crops
		16.4.1 CRISPR/Cas-Based Engineering of Crops for Virus Resistance
		16.4.2 CRISPR/Cas-Based Genetic Modification of Plants Against Fungal Disease
		16.4.3 Bacterial Resistance Achieved Through CRISPR/Cas9
	16.5 Tolerance to Herbicides and Abiotic Stress Factors via CRISPR/Cas9
	16.6 Improvement of Crop Yield, Nutritional Quality and Storage Using CRISPR/Cas9
	16.7 Conclusion
	References
17 Utilizing RNA-Based Approaches to Understand Plant-Insect Interactions
	17.1 Introduction
	17.2 Revolution in RNA Silencing
	17.3 siRNA and miRNA
	17.4 siRNA
	17.5 miRNAs
	17.6 Factors Affecting the Efficiency of RNAi
	17.7 Targeted Nucleotide Sequence
	17.8 dsRNA Length and Concentration
	17.9 Life Stages of Targeted Insect
	17.10 Target Gene
	17.11 Ingestion of dsRNAs Potential for Pest Management
	17.12 dsRNA Inside Insect Gut
	17.13 Transformative Versus Non-transformative RNAi
	17.14 Transformative RNAi
	17.15 Non-transformative RNAi
	17.16 Use of CRISPR Technologies in Understanding Plant-Insect Interactions
		17.16.1 Introduction to CRISPR
		17.16.2 Classification of CRISPR
		17.16.3 Utilizing the CRISPR/Cas9 Technology in Modulating Plants-Insects Interactions
	17.17 General Lab Practices in Gene Editing Plants and Insects
	17.18 Transgene-Free Approaches
	17.19 Risk Assessment of Release of Gene Edited Insects
	17.20 Use of Other CRISPR/Cas13 System in Insect-Pest Management in Agriculture
	17.21 Future Prospective with Cas3, Cas12, Cas14 Systems and Cas9 Variants in Pest Management
	17.22 Programmable Base Editing in Insect-Pest Management
	17.23 Latest Use of Leaper Technology in RNA Editing
	17.24 Conclusions and Perspectives
	References