This book discusses CRISPR/Cas- one of the most powerful tools available to scientists for genome editing. CRISPR/Cas is not only a genome editing tool, but researchers have also engineered it for gene regulation, genome imaging, base editing and epigenome regulations. This book describes the entire toolkit for CRISPR/Cas. The opening section gives an introduction to the technique and compares it with other genome editing tools. Further section gives a historical perspective of the tool, along with its detailed classification. The next chapters describe bioinformatic tools in CRISPR/Cas, and delivery methods for CRISPR/Cas. The book also discusses about the applications of CRISPR/Cas beyond genome editing and use of CRISPR for rewriting genetic codes. The book dedicates a section to the use of CRISPR in plants. The book culminates with a chapter on the current status, challenges and shortcomings of the CRISPR/Cas genome editing tool.
The book would be highly interesting to students and researchers in molecular biology, biochemistry, biotechnology, food science, agriculture and plant sciences.
Author(s): Aftab Ahmad, Sultan Habibullah Khan, Zulqurnain Khan
Publisher: Springer
Year: 2022
Language: English
Pages: 333
City: Singapore
Foreword
Preface
Contents
About the Editors
1: An Introduction to Genome Editing Techniques
1.1 What Is Genome Editing?
1.2 History of Genome Editing
1.3 Different Techniques of Genome Editing
1.3.1 Cre-LoxP System
1.3.2 Meganucleases
1.3.3 Zinc Finger Nucleases (ZFNs)
1.3.4 TALENs
1.3.5 CRISPR/Cas System
1.3.5.1 Mechanism of Action of CRISPR/Cas
1.4 Rise of CRISPR/Cas
1.5 Comparison of CRISPR/Cas with Other Genome Editing Tools
References
2: History and Classification of CRISPR/Cas System
2.1 Introduction of CRISPR/Cas System
2.2 History of CRISPR/Cas
2.3 Classification of CRISPR/Cas System
2.4 Types of CRISPR
2.4.1 CRISPR/Cas Type I
2.4.2 CRISPR/Cas Type II
2.4.3 CRISPR/Cas Type III
2.4.4 Other Variants
2.5 CRISPR/Cas System for DNA Editing
2.6 CRISPR/Cas for RNA Editing
2.7 Future Prospects and Final Remarks
References
3: Bioinformatic Tools in CRISPR/Cas Platform
3.1 Introduction
3.2 Fundamentals of CRISPR/Cas Experiment and sgRNA Design
3.2.1 Good Gene Annotation: An Essential Requirement
3.2.2 Different Guidelines for Different Applications
3.2.3 Best Design Linked with Availability of More Data
3.3 sgRNA Design Process: An Overview
3.3.1 Selection of Desired Genetic Modification
3.3.2 Choice of Appropriate Expression System
3.3.3 Selection of Appropriate Cas Endonuclease
3.3.4 Selection of Gene or Genetic Element
3.3.5 Searching of Target Site for Intended Gene Modification
3.3.6 Sequencing of Target Site and Design of sgRNA
3.3.7 Selection of Suitable gRNA
3.3.8 Design Criteria for Genome-Wide CRISPR Libraries
3.4 Specificity in CRISPR/Cas
3.4.1 Alignment-Based Approach to Predict Specificity
3.4.2 Specificity Prediction Through Scoring-Based Tools
3.4.2.1 Hypothesis-Driven Methods
3.4.2.2 Learning-Based Methods
3.5 Factors Affecting Specificity
3.5.1 Importance of PAM in CRISPR/Cas Specificity
3.5.2 Seed Sequence of sgRNA
3.5.3 Effective Concentration of Cas9/sgRNA Complex
3.5.4 Importance of sgRNA Sequence
3.5.4.1 Chromatin Accessibility and Epigenetic Features Affecting Binding of Cas
3.5.4.2 Numbers of Seed Sequence in the Genome
3.5.4.3 Length of Target Sequence Influences Specificity
3.5.5 sgRNA Scaffold
3.5.6 Repair Outcomes of DSBs
3.6 Efficiency of sgRNA
3.7 Off-Targeting in CRISPR/Cas
3.8 Application-Specific Design of sgRNA
3.8.1 sgRNA for KO Applications
3.8.2 Position of sgRNA for KI Applications
3.8.3 Designing sgRNA for CRISPRi and CRISPRa
3.8.4 SgRNA in Epigenetic Regulation
3.8.5 Design Criteria for Base Editing
3.8.6 Designing sgRNA for RNA Editing
3.9 Design Tools for sgRNA
3.9.1 CHOPCHOP
3.9.2 Base Editing (BE)-Analyzer and BE-Designer
3.9.3 CRISPOR
3.9.4 CRISFlash
3.10 Prospects
Appendix 1: List of Useful Bioinformatics Tools and Databases for Gene Modification Research
Appendix 2: List of Commercial and Non-profit Sources of CRISPR/Cas Reagents
References
4: Delivery Methods for CRISPR/Cas Reagents
4.1 Introduction
4.2 CRISPR/Cas reagents
4.2.1 DNA
4.2.2 mRNA
4.2.3 Ribonucleoproteins (RNPs)
4.3 Delivery Approaches in CRISPR/Cas
4.3.1 Viral Delivery Methods
4.3.1.1 Adenoviruses (AdVs)
4.3.1.2 Adeno-Associated Viruses (AAVs)
4.3.1.3 Lentiviral Vectors (LVs)
4.3.1.4 Bacteriophages
4.3.2 Non-viral Delivery Methods
4.3.2.1 Polymeric Materials
4.3.2.2 Lipid Nanoparticles
4.3.2.3 Cell-Penetrating Peptides (CPPs)
4.3.2.4 Gold Nanoparticles (AuNPs)
4.3.3 Physical Methods
4.3.3.1 Microinjection (MI)
4.3.3.2 Electroporation (EP)
4.3.3.3 Hydrodynamic Injection (HDI)
4.3.3.4 Gesicles
4.3.3.5 iTOP
4.3.3.6 Mechanical Cell Deformation
4.3.4 Potential Future Delivery Tools
4.3.4.1 Inorganic Nanoparticles
4.3.4.2 Lipid-Coated Mesoporous Silica Particles (LC-MSPs)
4.3.4.3 Streptolysin O (SLO)
4.3.4.4 Multifunctional Envelope-Type NanoDevices (MENDs)
4.4 Delivery Methods in Plants
4.4.1 Direct Methods
4.4.2 Indirect Methods
4.4.2.1 Agrobacterium
4.4.2.2 Virus-Mediated Delivery of CRISPR Reagents
4.5 Challenges and Future Prospect
References
5: Reengineering of the CRISPR/Cas System
5.1 Generating Knockout and Knock-In with CRISPR/Cas9
5.2 Increasing Specificity of Cas Protein
5.2.1 Guide RNA (gRNA) Selection Through Computational Approaches and Predictive Models
5.2.2 Cas9 Orthologs and Variants
5.2.3 Protein Engineering Manifest PAMs
5.2.4 Improvement in Cas9 Specificity Via dCas9
5.2.5 Other Modifications to Increase Specificity of Cas Protein
5.3 Reducing off-Target
5.3.1 CRISPR/Cas9 and Methods to Reduce off-Target Mutations
5.3.2 Methods for Off-Target Detection
5.3.2.1 Computational Method
5.3.2.2 Cell-Based Assays
5.3.2.3 In Vitro Assay
5.4 Expanding PAM Requirements
5.5 CRISPR/Cas12 for Editing Genome
5.5.1 Application
5.6 Genome Editing Through CasX
5.6.1 Applications of CasX
5.6.1.1 Human and E. coli Genome Editing
5.6.1.2 Other Gene Editing Proteins
5.7 Multiplex Genome Editing Through CRISPR/Cas
5.7.1 Multiplex Genome Editing
5.7.2 Applications
5.8 Future Perspectives
References
6: Beyond Genome Editing: CRISPR Approaches
6.1 Introduction
6.2 Genome Editing with CRISPR/Cas
6.2.1 Prominent CRISPR/Cas Systems
6.3 CRISPRi and CRISPRa for Transcriptional Regulation of Genes
6.4 Epigenome Modulation Through CRISPR
6.5 CRISPR-Mediated Site-Specific Base Editing
6.6 Prime Editing (PE)
6.7 Genome-Wide Screening
6.8 Genome Imaging with CRISPR/Cas
6.9 Tracking Cell Lineage Through CRISPR/Cas
6.10 CRISPR-Based Gene Drives
6.11 CRISPR-Based Directed Evolution (CDE)
6.12 CRISPR/Cas13 for RNA Editing
6.13 Prospects
References
7: RNA Editing with CRISPR/Cas13
7.1 Introduction
7.2 RNA Editing Systems: An Overview
7.2.1 RNA Interference (RNAi)
7.2.2 Type II CRISPR/Cas Systems for RNA Editing
7.2.2.1 RCas9 System for Targeting RNA
7.2.2.2 FnCRISPR/Cas9 System for RNA Editing
7.2.3 Type III CRISPR/Cas System (CRISPR/Cas3)
7.2.3.1 Types of Type III CRISPR/Cas System
7.2.3.2 RNP Complex and Processing of crRNA
7.2.3.3 Nuclease Activities of Type III System
7.2.3.4 Sequence-Specific RNA Cleavage
7.2.3.5 Non-specific Cleavage of ssDNA
7.2.3.6 Non-specific RNA Cleavage
7.2.4 Type VI CRISPR/Cas Systems (CRISPR/Cas13)
7.3 Structure of crRNA in CRISPR/Cas13 Systems
7.3.1 Type VI-A (Cas13a)
7.3.2 Type VI-B (Cas13b)
7.3.3 Type VI-C (Cas13c) and Type VI-D (Cas13d)
7.4 Processing of Pre-crRNA
7.4.1 Cas13a
7.4.2 Cas13b
7.4.3 Cas13c and Cas13d
7.4.4 Processing of crRNA-Spacer in Type VI Systems
7.4.5 crRNA-Spacer Organization and Target Search by Cas13
7.4.6 The Post-Flanking Sequence (PFS)
7.4.7 Collateral Cleavage/Global RNA Degradation
7.4.8 Specificity of Cas13
7.5 Comparison of CRISPR/Cas13 with Other RNA Editing Systems
7.5.1 CRISPR/Cas13 Vs. RNAi
7.5.2 CRISPR/Cas13 Vs. CRISPR/Cas9
7.6 Types of Cas13 Effector Nuclease
7.6.1 Type VI-A (CRISPR/Cas13a)
7.6.2 Type VI-B1 and B2 (CRISPR/Cas13b)
7.6.3 Type VI-C (CRISPR/Cas13c)
7.6.4 Type VI-D (CRISPR/Cas13d)
7.6.5 Type VI-E (Cas13e) and VI-F (Cas13f)
7.7 Applications of CRISPR/Cas in RNA Editing
7.7.1 SHERLOCK: A CRISPR/Cas13-Based Viral Diagnostic Tool
7.7.2 CARVER
7.7.2.1 A Cas13-Based Detection and Prevention Platform Against RNA Viruses
7.7.3 CRISPR/Cas13-Based Antiviral Strategy Against COVID-19 (PAC-MAN)
7.7.4 Knockdown of RNA
7.7.5 RNA Editing with CRISPR/Cas13
7.7.6 Virus Interference
7.7.7 RNA Tracking and Imaging
7.7.8 Alternate Splicing
7.7.9 Regulation of Translation Through CRISPR/Cas13
7.7.10 Programmed Cell Death and CRISPR/Cas13
7.7.11 Processing of mRNA for Stress Tolerance in Plants
7.7.12 Gene Regulation with CRISPR/Cas
7.7.13 Isolation of Specific RNA
7.8 Future Prospect
References
8: CRISPR for Rewriting Genetic Code
8.1 Introduction
8.1.1 History of Rewriting Genetic Code
8.1.2 The Upper Hand of CRISPR on Other ``Genetic Code Writing Techniques´´
8.2 Multiplex Automated Genome Editing (MAGE)
8.2.1 Ways of Delivery
8.2.2 Applications
8.2.3 Challenges
8.3 Conjugative Assembly Genome Engineering (CAGE)
8.3.1 Applications
8.3.2 Challenges
8.4 Targeted Mutagenesis with CRISPR-Guided DNA Polymerase (EvolvR)
8.4.1 Applications
8.4.2 Challenges
8.5 Comparison of EvolvR with Other Genome Editing Methods
8.6 Ethical, Legal, and Social Considerations in Rewriting Genetic Codes Using CRISPR
8.7 Conclusions
References
9: Applications of CRISPR/Cas System in Plants
9.1 Introduction
9.2 Applications of CRISPR/Cas in Model Plants
9.3 Application of CRISPR Crops
9.4 Applications in Horticultural Crops
9.5 CRISPR/Cas9-Mediated Genome Editing in Legumes
9.6 CRISPR-Based Editing in Tree Plants
9.7 Biosafety and CRISPR-Edited Plants
9.7.1 Biosafety Concerns
9.8 Future Prospects of CRISPR/Cas in Plant Genome Engineering
References
10: Challenges and Future Prospects of CRISPR Technology
10.1 CRISPR/Cas and off-Targeting
10.1.1 Off-Targeting Minimization Strategies
10.1.2 CRISPR Tools for Minimizing off-Targets
10.2 Specificity Problem with CRISPR/Cas
10.2.1 dCas9 Specificity
10.2.2 Broader Implications of Specificity
10.3 Comparison with Other Genome Editing Tools
10.4 Choice of Delivery Methods
10.5 Ethical Issues and Public Acceptance
10.6 Future Prospect of CRISPR/Cas
References
