Foreword	5
	Contents	6
	About the Authors	8
	Part I: Introduction to the CRISPR Revolution	9
	Chapter 1: Viral Vectors, Engineered Cells and the CRISPR Revolution	10
	1.1 Introduction	11
	1.2 CRISPR-Cas Genome Manipulation	12
	1.2.1 A Brief Overview of Genome Modification Using Endonucleases	12
	1.2.2 CRISPR-Cas Systems	14
	1.2.3 CRISPR Tools in Biology	14
	1.3 Gene Therapy Using Viruses	15
	1.3.1 Retroviral Vectors	15
	1.3.2 Modifications and Implementation of Retroviral Vectors	16
	1.3.3 Translational and Clinical Progress Using Retroviral Vectors	17
	1.4 Adenoviral Vectors	17
	1.4.1 Modifications and Implementation of Adenoviral Vectors	18
	1.4.2 Translational and Clinical Progress Using Adenoviral Vectors	19
	1.5 Adenoviral-Associated Viral Vectors	20
	1.5.1 Modifications and Implementation of Adeno-associated Viral Vectors	21
	1.5.2 Translational and Clinical Progress Using Adeno-­associated Viral Vectors	22
	1.6 Ex Vivo CRISPR Therapies	23
	1.6.1 CAR T-Cell Therapy	23
	1.6.2 iPSCs	25
	1.7 Conclusion	27
	References	27
	Chapter 2: Combining Engineered Nucleases with Adeno-­associated Viral Vectors for Therapeutic Gene Editing	35
	2.1 Introduction	36
	2.2 Therapeutic Gene Editing	37
	2.2.1 AAV	40
	2.2.2 Nucleases and AAV for Therapeutic Gene Editing	41
	2.2.3 Challenges	42
	2.3 Conclusion	45
	References	45
	Part II: CRISPR in Model Systems	49
	Chapter 3: From Reductionism to Holism: Toward a More Complete View of Development Through Genome Engineering	50
	3.1 Introduction and Historical Context	50
	3.2 CRISPR Genome Editing in Brief	53
	3.3 A Genomics Perspective	54
	3.3.1 Gene Network Analysis with CRISPR GE	55
	3.3.2 Mapping and Understanding Regulatory DNA Within the Genomic Context with CRISPR GE	57
	3.4 An Epigenomics Perspective	60
	3.4.1 Manipulating DNA and Histone Modifications with CRISPR GE	61
	3.4.2 Tracking 3D Genomic Structure with CRISPR GE	63
	3.5 A Cellular Perspective	65
	3.5.1 Lineage Tracing with CRISPR GE	66
	3.5.2 CRISPR GE and Ex Vivo Organogenesis	69
	3.5.3 Final Thoughts	71
	References	71
	Chapter 4: A Transgenic Core Facility’s Experience in Genome Editing Revolution	80
	4.1 Pronuclear Microinjection	81
	4.2 ES Cell Injection	82
	4.3 Cytoplasmic Microinjection	82
	4.4 A New Era of Animal Model Production by the CRISPR/Cas9 Technology	83
	4.5 Guide RNA Activity is the Key	84
	4.6 Design of the CRISPR/Cas9-Mediated Targeting	85
	4.7 Conclusion and Perspectives	91
	References	92
	Chapter 5: Genome Editing to Study Ca2+ Homeostasis in Zebrafish Cone Photoreceptors	96
	5.1 Introduction	96
	5.2 Ca2+ Homeostasis in Photoreceptors	97
	5.3 Genome Editing in Zebrafish	99
	References	102
	Chapter 6: CRISPR: From Prokaryotic Immune Systems to Plant Genome Editing Tools	106
	6.1 Introduction	107
	6.2 What Is CRISPR?	108
	6.3 History of CRISPR	110
	6.4 Cas9 and Cpf1: The Lead Players in CRISPR-Based Genome Editing	111
	6.5 Modification of the CRISPR System in Plants	114
	6.6 Application of CRISPR in Plants	116
	6.6.1 Model Plants	116
	6.6.2 Application in Crop Plants	119
	6.6.3 Off-Target Effects	120
	6.7 Future Prospects of Genome Editing in Plants	120
	References	122
	Part III: The Future of CRISPR	126
	Chapter 7: Target Discovery for Precision Medicine Using High-Throughput Genome Engineering	127
	7.1 Introduction	127
	7.2 Technologies for CRISPR Screens	128
	7.2.1 From Gene Editing to Pooled Screens	128
	7.2.2 Types of CRISPR Screens	135
	7.3 CRISPR Screen Applications: Genetic Mechanisms of Human Disease and Therapeutic Development	137
	7.3.1 CRISPR Screens in Cancer for Synthetic Lethality and Drug Resistance	137
	7.3.1.1 Identifying Cancer-Specific Vulnerabilities	137
	7.3.1.2 Understanding Mechanisms of Drug Resistance	138
	7.3.1.3 Examining Noncoding Regulators of Cancer Gene Expression	139
	7.3.2 CRISPR Screens in Infectious Disease	140
	7.3.3 CRISPR Screens for Understanding and Treating Inborn Genetic Disorders	141
	7.4 Conclusion and Future Perspectives	143
	References	143
	Chapter 8: CRISPR in the Retina: Evaluation of Future Potential	150
	8.1 Introduction: CRISPR and the Retina	151
	8.2 “Is There Anything You Can Do for Me?”	152
	8.3 Research Highlights	153
	8.3.1 Limitations and Imprecision Medicine	153
	8.3.2 Future Strategies	154
	References	155
	Chapter 9: The Future of CRISPR Applications in the Lab, the Clinic and Society	159
	9.1 The Scientific Community Debates Somatic and Germline Genome Modification	161
	9.2 The Somatic-Germ Line Barrier	163
	9.3 A New Paradigm for Genomic Medicine	166
	9.4 Converging Technologies to Democratize CRISPR	166
	9.5 The Impact of CRISPR on Human Biodiversity	168
	9.6 Editing the Ecosystem	170
	9.7 CRISPR Calls for a Conversation	171
	References	174