Precision Medicine, CRISPR, and Genome Engineering (eBook)

For years, Stephen Tsang has been culturing embryonic stem (ES) cells and created the first mouse model for a recessive form of retinitis pigmentosa by applying homologous recombination to ES cell technology. Two elements define his laboratory. First, by leveraging his genetics clinical practice, in which over 1000 retinal patients are cared for, he brings an array of clinical resources to his research, including stem cells and live imaging data. Second, he and his students are recognized authorities in a broad array of state-of-the-art technologies. Most recently, he was invited as a Moderator for the Gene Editing/Rewriting the Genome session during the 65th American Society of Human Genetics Annual Meeting.

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