Genome Engineering Via Crispr-cas9 System

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-containing ``hotspots''
			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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