Genome Engineering Via Crispr-cas9 System

✍ Scribed by Vijai Singh (editor), Pawan Kumar Dhar (editor)

• Publisher: Academic Pr
• Year: 2020
• Tongue: English
• Leaves: 357
• Edition: 1
• Category: Library

✦ Synopsis

Genome Engineering via CRISPR-Cas9 Systems presents a compilation of chapters from eminent scientists from across the globe who have established expertise in working with CRISPR-Cas9 systems. Currently, targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative simplicity to which they can be designed, used and applied. However, it is not easy to find relevant information gathered in a single source. The book contains a wide range of applications of CRISPR in research of bacteria, virus, algae, plant and mammalian and also discusses the modeling of drosophila, zebra fish and protozoan, among others.

Other topics covered include diagnosis, sensor and therapeutic applications, as well as ethical and regulatory issues. This book is a valuable source not only for beginners in genome engineering, but also researchers, clinicians, stakeholders, policy makers, and practitioners interested in the potential of CRISPR-Cas9 in several fields.

• Provides basic understanding and a clear picture on how to design, use and implement the CRISPR-Cas9 system in different organisms
• Explains how to create an animal model for disease research and screening purposes using CRISPR
• Discusses the application of CRISPR-Cas9 systems in basic sciences, biomedicine, virology, bacteriology, molecular biology, neurology, cancer, industry, and many more

✦ Table of Contents

Genome Engineering via CRISPR-Cas9 System
Copyright
Dedication
Contributors
About the editors
Foreword
Preface
Acknowledgments
Vijai Singh
Pawan K. Dhar
1. An introduction to genome editing CRISPR-Cas systems
1.1 Introduction
1.2 History and classification of CRISPR-Cas systems
1.3 Milestones in the CRISPR-Cas systems
1.4 Development of CRISPR-CAS9 system for genome editing
1.4.1 Microbial genome editing using CRISPR-Cas9 system
1.4.2 Viral genome editing using CRISPR-Cas9 system
1.4.3 Mammalian cells genome editing using CRISPR-Cas9 system for therapeutic applications
1.4.3.1 Cancer therapy
1.4.3.2 Duchenne muscular dystrophy therapy
1.4.3.3 Beta-thalassemia therapy
1.4.3.4 Blindness therapy
1.4.3.5 Cardiovascular disease therapy
1.5 Recent developments in CRISPR interference platform
1.5.1 CRISPRi
1.5.2 CRISPRa
1.5.3 Loci imaging
1.6 Conclusion and future remarks
References
2. Evolution and molecular mechanism of CRISPR/Cas9 systems
2.1 Introduction
2.2 Evolution of CRISPR/Cas9 systems
2.3 Classification of CRISPR/Cas systems
2.3.1 Class 1 systems
2.3.2 Class 2 systems
2.4 Molecular mechanism of CRISPR/Cas-mediated defense systems
2.4.1 Acquisition of new spacer
2.4.2 Processing of CRISPR array
2.4.3 CRISPR-interference
2.5 Application of CRISPR/Cas9 systems
2.6 Conclusions
References
3. Exploring the potential of CRISPR-Cas9 for the removal of human viruses
3.1 Introduction
3.2 CRISPR-Cas9 system as an antiviral agent
3.2.1 Human immunodeficiency virus
3.2.2 Hepatitis B virus
3.2.3 Epstein-Barr virus
3.2.4 Herpes simplex virus
3.2.5 Human papillomavirus
3.3 CRISPR delivery in mammalian cells
3.4 Challenges to the use of CRISPR-CAS9 as therapy
3.5 Conclusion and future perspective
References
4. Programmable removal of bacterial pathogens using CRISPR-Cas9 system
4.1 Introduction
4.2 Mechanism of CRISPR-Cas systems
4.3 Application of CRISPR-Cas9 system as an antimicrobial agent
4.3.1 CRISPR-Cas9 for removal of mammalian pathogenic bacteria
4.3.2 CRISPR-Cas9 for removal of plant pathogenic bacteria
4.4 Bacteriophage engineering to extend the host range
4.5 Conclusion and future remarks
Acknowledgment
References
5. Targeted genome editing using CRISPR/Cas9 system in fungi
5.1 Introduction
5.2 Genome editing in yeasts
5.2.1 Genome editing in S. Cerevisiae
5.2.1.1 Expression and delivery of Cas9 and sgRNA
5.2.1.2 Targeted gene modification
5.2.1.3 Multiplex genome editing and gene integration
5.2.1.4 Chromosomal engineering
5.2.1.5 High-throughput genome wide analyses
5.2.1.6 Precise base editing
5.2.2 Genome editing in non-conventional yeasts
5.2.2.1 Genome editing in S. pombe
5.2.2.2 Genome editing in Y. lipolytica
5.2.2.3 Genome editing in P. pastoris
5.2.2.4 Genome editing in K. lactis and Kluyveromyces marxianus
5.2.2.5 Genome editing in Saccharomyces pastorianus
5.2.2.6 Genome editing in O. polymorpha and Ogataea parapolymorpha
5.2.2.7 Genome editing in pathogenic yeasts
5.2.3 Conclusion, challenges, and future remarks
5.3 Genome editing in filamentous fungi
5.3.1 Genome editing in filamentous fungi in 2015
5.3.2 Genome editing in filamentous fungi after 2016
5.3.3 Large deletion of some filamentous fungi in genome editing via NHEJ repair
5.3.4 Conclusion and future remarks
References
6. CRISPR-Cas9 system for fungi genome engineering toward industrial applications
6.1 Introduction
6.2 Challenges in editing fungal genome
6.3 Industrial applications of CRISPR-Cas9 methods in fungi genome editing
6.4 Implementations of the CRISPR-Cas9 in fungi
6.5 CRISPR-based gene regulation in fungi
6.6 CRISPR-Cas9 a novel approach for biological control
6.7 Further developments required for fungi genome editing
6.8 Conclusion and future prospects
Acknowledgment
References
7. Development and challenges of using CRISPR-Cas9 system in mammalians
7.1 Introduction
7.2 Principle mechanism behind CRISPR-Cas9 mediated gene editing
7.3 Delivery of CRISPR-Cas9 component
7.4 Recent development and applications of CRISPR-Cas9 for human and mammalian diseases
7.4.1 Cancer
7.4.2 Cataract
7.4.3 Duchenne muscular dystrophy
7.4.4 Tyrosinemia type-1
7.4.5 Cystic fibrosis
7.4.6 Urea cycle disorder
7.4.7 Blood disorder
7.4.8 Retinal degenerative
7.4.9 Cardiovascular disease
7.4.10 Amyotrophic lateral sclerosis
7.4.11 Huntington's disease
7.5 Key issues and challenges
7.6 Conclusions and future remarks
Acknowledgment
References
8. CRISPR-Cas9 system a mighty player in cancer therapy'' 8.1 Introduction 8.2 Functional characterization of cancer-related genes by conventional methods 8.3 Involvements of the non-coding region of the human genome in a certain type of cancers could give a novel therapeutic targets 8.3.1 Long non-coding RNA (lncRNAs) functional drivers of breast cancer progression and invagination 8.4 Challenges and advancement needed in CRISPR-Cas9 method for cancer treatments 8.5 CRISPR-Cas9 and the future of cancer therapy References 9. CRISPR-Cas9 for therapy: the challenges and ways to overcome them 9.1 Introduction 9.2 CRISPR-Cas9 as a drug 9.3 A match made in heaven; iPSC and CRISPR-Cas9 9.4 Ex-vivo versus in-vivo editing 9.5 Bench-to-bedside challenges 9.6 Conclusion References 10. Engineering of Cas9 for improved functionality 10.1 Introduction 10.2 Cas9 variants with altered nuclease activity 10.2.1 nCas9 10.2.2 Dead Cas9 (dCas9) 10.3 Cas9 variants with improved PAM specificity 10.4 Switchable Cas9 10.4.1 Reconstitution of Cas9 via sgRNA 10.4.2 Ligand-dependent re-assembly of Cas9 10.4.3 Photo-inducible reconstitution of Cas9 10.4.4 Intein-inducible recombining of Cas9 10.5 Inducible Cas9 10.6 gRNA editing 10.7 Other CRISPR-associated endonucleases References 11. The current progress of CRISPR/Cas9 development in plants 11.1 Introduction 11.2 Mechanism of Crispr/Cas9 11.3 Multiplex Crispr/Cas9 11.4 Metabolic engineering in plants using Crispr/Cas9 11.5 Crispr/Cas9 mediated live cell imaging 11.6 Non-transgenic plants through CRISPR/Cas9 11.7 Conclusions and future remarks Acknowledgments References 12. Fruit crops improvement using CRISPR/Cas9 system 12.1 Introduction 12.2 Nutritional aspects of fruit crops 12.3 Genome editing as a tool towards fruit crop improvement 12.3.1 CRISPR/Cas9 technology toward nutritional enrichment of fruit crop 12.3.1.1 Gene silencing/knockout using CRISPR/Cas9 12.3.1.2 Gene knock-in or promoter activation using CRISPR/Cas9 12.4 Crispr/Cas9 system: a tool for improving stress tolerance in fruit crops 12.4.1 Abiotic stress 12.4.2 Biotic stress 12.5 Challenges pertaining to fruit crop improvement via Crispr/Cas9 technology 12.5.1 Genome completeness and off-target effects 12.5.2 Tissue culture and CRISPR/Cas9 delivery methods 12.5.3 Challenges in CRISPR/Cas9 mediated knock-in approach 12.6 Conclusion and future perspective Acknowledgments References 13. CRISPR/Cas9 engineered viral immunity in plants 13.1 Introduction 13.2 Plant viruses and existing virus control strategies 13.3 Gene editing with CRISPR-Cas system 13.4 CRISPR-Cas-mediated viral resistance through PDR approach 13.5 CRISPR-Cas mediated viral resistance by interfering host encoded genes 13.6 Conclusion and future perspectives References 14. Genome engineering in medicinally important plants using CRISPR/Cas9 tool 14.1 Introduction 14.2 Designing of gRNA and vectors construction 14.3 CRISPR-Cas9 construct delivery methods into plant cells 14.3.1 Agro-infiltration into leaf 14.3.2 A. rhizogenes-mediated transformation 14.3.3 Biolistic transformation facilitate for DNA free editing 14.3.4 PEG mediated delivery of CRISPR-Cas9 ribonucleoprotein complexes 14.4 Pathway engineering using CRISPR-Cas9 14.5 Editing in hairy roots of medicinal plants for producing secondary metabolites 14.6 Future perspective of genome editing in medicinal plants References 15. Genome editing of algal species by CRISPR Cas9 for biofuels 15.1 Introduction 15.2 Genetic engineering of algae 15.2.1 Methods of DNA delivery 15.2.2 Selectable marker 15.2.3 Merits and demerits of nuclear and chloroplast transformation 15.3 CRISPR 15.3.1 Brief introduction to CRISPR-Cas9 15.3.2 Mechanism of CRISPR-Cas9 guided cleavage 15.3.3 Cas9 variants 15.3.4 Modes of Cas9 expression into the algal systems 15.3.4.1 Vector-based expression 15.3.4.2 RNP 15.3.4.3 mRNA 15.4 CRISPR over random mutagenesis and RNAi 15.5 Target pathways for development of microalgae as biofuel feedstocks 15.6 CRISPR-Cas9 in microalgae 15.6.1 Genome editing in C. reinhardtii using Cas9 nuclease 15.6.2 Demonstration of Cas9 suitability in diatom species by targeting genes with visible or auxotrophic phenotypes 15.6.3 CRISPR/Cas9 as a tool of choice for editing in the industrial microalgae 15.6.3.1 Genome editing in Nannochloropsis 15.6.3.2 Proof of concept study in Coccomyxa sp 15.6.4 Use of Cpf1 and other Cas9 homologs in algae for DNA integration 15.6.5 Episomes for DNA editing 15.6.6 Biolistic delivery of RNPs to generate auxotrophic mutants in diatom 15.7 CRISPR workflow in microalgae 15.7.1 Selection of target gene(s) 15.7.2 Choice of Cas9 expression system 15.7.3 sgRNA design 15.7.4 Transformation of cells with Cas9 15.7.5 Screening of mutants for editing 15.7.6 Characterization of mutants 15.8 Conclusion, challenges and future remarks References 16. Development and use of CRISPR in industrial applications 16.1 Historical perspectives 16.1.1 Genome editing strategies 16.1.2 Discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 16.1.3 Mechanism of action of CRISPR-Cas immune systems 16.1.4 The rise of CRISPR 16.2 Development of CRISPR based technologies 16.3 Design tools for CRISPR-Cas9 based genome editing 16.4 Industrial products developed using CRISPR 16.4.1 Biofuels 16.4.2 Organic acids 16.4.3 Phytochemicals 16.4.4 Polyhydroxyalkanoates (PHA) 16.4.5 Amino acids 16.4.6 Health care 16.4.7 Engineering sugar metabolism for improved feedstock utilization 16.5 Conclusion References 17. Functional understanding of CRISPR interference: its advantages and limitations for gene silencing in bacteria 17.1 Introduction 17.1.1 Need of genome editing in bacteria: annotating the unknowns 17.1.2 CRISPR-Cas system: the foremost choice of genome editing in the 21st century 17.1.3 Type II CRISPR-Cas system: a role model for CRISPR-interference 17.2 Gene silencing by CRISPRi in bacteria 17.2.1 The concept of CRISPRi 17.2.2 Critical components of CRISPRi 17.2.2.1 The genesis of dCas 17.2.2.2 The crRNA and the tracrRNA 17.2.2.3 Protospacer adjacent motif (PAM) 17.2.3 Understanding the functional complex formation 17.2.3.1 Formation of active DNA surveillance complex 17.2.3.2 Surveillance of PAM sequence 17.2.3.3 Establishment of dCas9-gRNA-DNA ternary complex 17.2.4 Knocking down the gene expression by CRISPRi in bacteria 17.2.4.1 Selection of promoter for expression of dcas9 and gRNA 17.2.4.2 Use of single or dual plasmid vectors for co-expression of dCas9 and gRNA 17.2.4.3 Selection of target site for hybridization of gRNA 17.2.4.4 Designing of gRNA: as stated above, the full length gRNA requires three distinct sequences 17.2.4.5 Evaluation of gene silencing 17.3 Advantages and limitations of CRISPRi in bacteria 17.3.1 Advantages of CRISPRi 17.3.1.1 Reversibility 17.3.1.2 Utility toward characterization of essential genes 17.3.1.3 Rapid characterization of multiple targets 17.3.1.4 In vivo characterization of critical protein motifs 17.3.1.5 Multiplexing 17.3.1.6 Identification of drug targets 17.3.1.7 Utility in mapping the promoter region and identification of operons 17.3.1.8 Other advantages 17.3.2 Limitations of CRISPRi 17.3.2.1 Requirement of codon-optimized Cas proteins for efficient expression 17.3.2.2 Optimization of gRNA 17.3.2.3 Requirement of PAM-containinghotspots''
17.3.2.4 Pleiotropic effect on adjacent genes
17.3.2.5 Consistent use of antibiotics
17.4 Concluding remarks
Acknowledgments
References
18. Genome engineering in insects: focus on the CRISPR/Cas9 system
18.1 Introduction
18.2 Tools used for genome engineering
18.2.1 ZFN
18.2.1.1 Mechanism of ZFN
18.2.1.2 Advantages and disadvantage of ZFN
18.2.2 TALEN
18.2.2.1 Mechanism of TALEN
18.2.2.2 Advantages and disadvantages of TALEN
18.2.3 CRISPR/Cas9
18.2.3.1 History of CRISPR/Cas9
18.2.3.2 Mechanism of CRISPR/Cas9
18.2.3.3 SgRNA
18.2.3.4 PAM
18.2.3.5 Advantage of CRISPR/Cas9 system
18.3 Genome engineering in insects using CRISPR/Cas9
18.3.1 Fruit fly (D. melanogaster)
18.3.2 Mosquitoes
18.3.3 Silkworm (B. mori)
18.3.4 Butterflies
18.4 Targeting efficiency and off-target effects of CRISPR/Cas9
18.5 Genome engineering in insects using ZFN
18.6 Genome engineering in insects using TALEN
18.7 Gene drive
18.7.1 History of gene drive systems
18.7.2 Mechanism of CRISPR/Cas9 gene drive
18.8 Ethical concerns of genome editing
18.9 Conclusion
Acknowledgments
References
19. Recent progress of CRISPR-Cas9 in zebra fish
19.1 Introduction
19.2 Methods of genome editing
19.2.1 Zinc finger proteins (ZFNs)
19.2.2 Transcription activator-like effector nucleases (TALEN)
19.2.3 CRISPR-Cas9 for genome editing
19.3 CRISPR-Cas9 system for genome editing of zebra fish
19.4 Applications of CRISPR-Cas9 in zebrafish
19.4.1 Use of CRISPR-Cas9 system in developmental biology
19.4.2 Use of CRISPR-Cas9 system in neuronal development
19.4.3 Gene therapy
19.4.4 Cardiomyopathies
19.5 Conclusion and future remarks
References
20. CRISPR: a revolutionary tool for genome engineering in the protozoan parasites
20.1 Introduction
20.2 Limitations in genome engineering of apicomplexan parasites
20.3 Gene editing tools for studying protozoan parasites
20.3.1 CRISPR/Cas9 for Plasmodium gene editing
20.3.2 CRISPR/Cas9 for T. gondii gene editing
20.3.3 CRISPR/Cas9 for C. parvum gene editing
20.4 Concluding remarks
Acknowledgments
References
21. Emergent challenges for CRISPR: biosafety, biosecurity, patenting, and regulatory issues
21.1 Introduction
21.2 Biosafety
21.2.1 Cancer risks through TP53/p53 dysfunction
21.2.2 Off-target effects
21.2.3 Genomic rearrangements and mosaicism
21.2.4 Human germ-line and embryo modification
21.2.5 Delivery of CRISPR products
21.2.6 Availability of CRISPR and DIY use
21.3 Biosecurity
21.3.1 The publicized severity of potential biosecurity threats
21.3.2 Realizing potential biosecurity threats
21.3.3 Gene drives and ecological disruption
21.3.4 Dual use technologies
21.3.5 Biological countermeasures against biosecurity risks
21.3.5.1 The DARPA Safe Genes project
21.3.5.2 CRISPR inhibitors
21.3.5.3 Gene drive resistance
21.4 Patenting CRISPR technologies and products
21.4.1 Obviousness in CRISPR patenting
21.4.2 Foundational patent dispute
21.4.3 The patenting of CRISPR products
21.4.3.1 Commercialization of CRISPR patents and the patent landscape
21.4.3.2 Surrogate licensing and exclusive licenses
21.4.3.3 Patent pooling
21.4.4 Patentability of CRISPR products
21.5 Regulatory issues with CRISPR products
21.5.1 The global regulatory landscape of CRISPR
21.5.2 Human gene editing
21.5.3 CRISPR in agricultural projects
21.5.4 Regulations around dual use and the sale of essential parts
21.5.5 Regulation through patents
21.6 Conclusions and future remarks
References
Appendices
Mathematical signs and symbols
List of abbreviations
Glossary
Author Index
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Subject Index
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