Targeted Genome Editing Using Site-Specific Nucleases (eBook)

ZFNs, TALENs, and the CRISPR/Cas9 System

Takashi Yamamoto (Herausgeber)

eBook Download: PDF

2015 | 2015
VIII, 205 Seiten
Springer Tokyo (Verlag)
978-4-431-55227-7 (ISBN)

Infos

This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases. Applications of genome editing tools are then described in detail, in iPS cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Tools that are mentioned include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, all of which have received much attention in recent years as breakthrough technologies. Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. Various genome modifications have proven successful, including gene knockout and knock-in experiments with targeting vectors and chromosomal editing. Genome editing technologies hold great promise for the future, for example in biomedical research, clinical medicine, and generation of crops and livestock with desirable traits. A wide range of readers will find this book interesting, and with its focus on applications in a variety of organisms and cells, the book will be valuable for life scientists in all fields.

Inhalt

Preface 6
Contents 8
Part I: Basics of Genome Editing 10
Chapter 1: Genome Editing Using Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) 11
1.1 Introduction 12
1.2 DSB Repair 14
1.3 ZFNs 14
1.3.1 Zinc-Finger Domain 14
1.3.2 FokI Nuclease Domain 15
1.3.3 Linkers 17
1.3.4 Engineering Methods of ZFs 17
1.3.4.1 Sequential Selection of ZFs with Novel Specificity Using a Randomized Library and a Phage Display 18
1.3.4.2 Bipartite Library and Two-Finger Archive 18
1.3.4.3 Modular Assembly 20
1.3.4.4 Bacterial-Based Selection of ZFs from a Randomized Library 20
1.3.4.5 Context-Dependent Assembly (CoDA) 21
1.3.5 Specificity of ZFNs 21
1.4 TALENs 22
1.4.1 Construction Methods for TALENs 24
1.4.1.1 Golden Gate Cloning-Based Assembly 24
1.4.1.2 Golden Gate PCR-Based Assembly 24
1.4.1.3 Restriction Enzyme and Ligation (REAL) Assembly 26
1.4.1.4 Fast Ligation-Based Automatable Solid-Phase High-Throughput (FLASH) System 26
1.4.2 Specificity of TALENs 26
1.5 Applications of ZFNs and TALENs 26
1.6 Summary 28
References 28
Chapter 2: CRISPR/Cas9: The Leading Edge of Genome Editing Technology 33
2.1 Introduction 33
2.2 The CRISPR/Cas System in Prokaryotic Adaptive Immunity 34
2.3 Application of CRISPR/Cas9 in Genome Editing 34
2.4 Targeting Specificity of CRISPR/Cas9 36
2.5 Double-Nicking and Dimeric FokI-dCas9 Strategies for Highly-Specific Genome Editing 37
2.6 Web-Based Software for Designing gRNA Targets and Predicting Off-Target Candidates 38
2.7 Construction of CRISPR/Cas9 Vectors 39
2.8 Methods for Introducing CRISPR/Cas9 into Cells and Organisms 40
2.9 Expanded Applications of CRISPR/Cas9 in Life Science Studies 41
Conclusions 43
References 43
Part II: Genome Editing in Cultured Cells and Various Organisms 50
Chapter 3: Editing Cultured Human Cells: From Cell Lines to iPS Cells 51
3.1 Target cells 51
3.1.1 Human Cell Lines 51
3.1.2 Genome Editing in Cultured Mammalian Cells 52
3.1.3 ES/iPS Cells as an Attractive Target 52
3.2 The Development of Engineered Nucleases 53
3.2.1 Meganuclease (Homing Nuclease) 53
3.2.2 ZFN (Zinc Finger Nuclease) 54
3.2.3 TALEN 56
3.2.4 CRISPR/Cas9 58
3.3 Delivery of Engineered Nucleases 59
3.3.1 Chemical Transfection 59
3.3.2 Physical Transfection 60
3.3.3 Viral Transfection 60
3.3.4 Bacterial Transfection 60
3.3.5 Protein Transfection 61
3.4 Genomic Manipulation Methods and Their Applications 61
3.4.1 Gene Knockout 61
3.4.2 Genome-Wide Loss-of-Function Libraries 61
3.4.3 Reporter Cell Lines 62
3.4.4 Knockin Transgenic Studies 62
3.4.5 Knockin Gene Therapy 63
3.4.6 Chromosomal Deletion or Inversion 63
3.5 Other Applications of Custom DNA Binding Modules 64
3.5.1 Controlling the Transcriptional Status 64
3.5.2 Modifying Epigenetic Marks 65
3.5.3 Optical Control of Transcription 66
3.5.4 Visualization for Live-Cell Imaging 66
3.5.5 Pull-Down of a Certain DNA Region by enChIP 66
3.6 Future Perspectives for Gene Therapy 67
Conclusions 67
References 68
Chapter 4: Genome Editing in Nematode 76
4.1 Introduction 76
4.2 Genome Engineering in Nematode Species Using ZFNs, TALENs, and CRISPR/Cas9 System 78
4.2.1 Targeted Knockout by ZFNs and Mos1 Transposon 78
4.2.2 Targeted Knockout Using TALEN and CRISPR/Cas9 System 78
4.2.3 Genome Editing by TALEN- and CRISPR-Mediated Homologous Recombination 81
4.2.4 Applications of Genome Editing Technology 82
Conclusion 83
References 84
Chapter 5: Highly Efficient Targeted Gene Disruption in the Silkworm, Bombyx mori, Using Genome Editing Tools 86
5.1 Introduction 86
5.2 Targeted Gene Disruption Using TALENs 88
5.3 Targeted Gene Disruption Using the CRISPR/Cas9 System 88
5.4 A Guideline for Gene Targeting Using Genome Editing Tools 90
5.4.1 Choice of Genome Editing Tools 90
5.4.2 Target Design 90
5.4.3 Construction of TALEN or gRNA Vectors 91
5.4.4 Mating Scheme 91
5.4.5 Common Problems 94
5.4.5.1 High Mortality in G0 Animals 94
5.4.5.2 Inter- and Intra-Strain Nucleotide Polymorphisms 94
5.4.5.3 Off-Target Effects 94
5.5 Further Applications of TALENs and CRISPR/Cas9 95
5.5.1 Large Deletions 95
5.5.2 Gene Knockin 95
5.5.3 Other Applications 96
5.6 Future Perspectives 96
References 97
Chapter 6: Genome Editing in Sea Urchin 102
6.1 Introduction on Sea Urchins 102
6.2 Disruption of Gene Functions 104
6.3 Use of Reporters to Visualize Gene Expression 107
6.4 Application of Genome Editing in Sea Urchin Research 109
References 109
Chapter 7: Genome Editing in Ascidians 112
7.1 Introduction: Experimental Techniques for Tunicates 112
7.1.1 Non-Ascidian Tunicates 112
7.1.2 Ascidians 114
7.2 Experimental Techniques in Ciona 115
7.3 Genome Editing in Ciona 117
7.3.1 ZFNs in Ciona 117
7.3.2 TALENs in Ciona 118
7.3.3 CRISPR/Cas9 in Ciona 119
7.3.4 Potential for Genome Editing in Future Ascidian Research 119
References 120
Chapter 8: Genome Editing in Zebrafish and Medaka 123
8.1 Introduction 124
8.2 Genome Editing Technologies in Fish 126
8.3 Multiple Genome Modifications in Zebrafish 130
8.4 Estimation of Deletion Pattern 131
8.5 Reduction of Off-Target Alteration 133
8.6 Other Applications of TALEN and CRISPR/Cas9 133
Conclusion 133
References 134
Chapter 9: Genome Editing Using Site-Specific Nucleases in Amphibians 136
9.1 Introduction 137
9.2 A Strategy for Genome Editing Using Site-Specific Nucleases in Amphibians 139
9.3 Targeted Gene Disruption Using ZFNs in Xenopus 141
9.4 Targeted Gene Disruption Using TALENs in Xenopus 142
9.5 Targeted Gene Disruption Using CRISPR/Cas9 in Xenopus 145
9.6 Targeted Gene Disruption Using TALENs and CRISPR/Cas9 in Urodeles 146
Concluding Remarks 148
References 150
Chapter 10: Genome Editing in Mice Using CRISPR/Cas 153
10.1 Introduction 154
10.2 Design and Validation of CRISPR/Cas9 System In Vitro 154
10.2.1 Initial Design and Validation Methods 154
10.2.2 Simpler and Faster Validation Method 155
10.3 Oocyte Injection 158
10.3.1 RNA Method 158
10.3.2 Plasmid Method 158
10.3.3 Discussion of RNA vs Plasmid 159
10.4 Oligo-Mediated Genetic Modifications 159
10.4.1 Point Mutations 159
10.4.2 Inserting Tags 161
10.5 dsDNA Mediated Homologous Recombination—Generation of Knockin Mice 161
10.5.1 Conditional Knockouts/Knockins 162
10.6 The Future of CRISPR/Cas9 Systems in Mice 163
10.6.1 Off-Target Mutations 163
10.6.2 Other Technologies 164
10.6.2.1 CRISPR Mediated Gene Activation/Inactivation 164
10.6.2.2 Observing the Genome 165
10.6.2.3 Gene Modification to Correct Impairment 165
Conclusion 166
References 167
Chapter 11: Genome Editing in Mice Using TALENs 169
11.1 Introduction 169
11.2 In Vivo Genome Editing in Mice 171
11.3 Knockout Mice 173
11.4 Knockin Mice 175
11.5 Off-Target Effects 177
11.6 Applications 178
References 181
Chapter 12: Engineered Nucleases Lead to Genome Editing Revolution in Rats 185
12.1 Genome Engineering Technologies in Rats 185
12.2 Gene Editing with ZFNs in Rats 187
12.3 Gene Editing with TALENs in Rats 189
12.4 Genome Editing with CRISPR/Cas9 in Rats 190
12.4.1 Knockout Rats with CRISPR/Cas9 190
12.4.2 Specificity of CRISPR to Targeted Sites 191
12.4.3 Knockin Rats with ssODNs 192
12.5 Advantages of Site-Specific Nuclease Technologies 192
Conclusions 194
References 194
Chapter 13: Genome Editing in Higher Plants 198
13.1 Introduction 198
13.2 Site-Directed Mutagenesis of Higher Plants Using Genome Editing Tools 200
13.2.1 Site-Directed Mutagenesis Using ZFNs 200
13.2.2 Site-Directed Mutagenesis Using TALENs 201
13.2.3 Site-Directed Mutagenesis Using CRISPR/Cas9 202
13.3 Gene Targeting and Targeted Gene Addition in Higher Plants Using Genome Editing Tools 203
13.4 Future Prospects 203
References 204

Produkt-Details

• Erscheint lt. Verlag: 5.1.2015
• Zusatzinfo: VIII, 205 p. 45 illus., 33 illus. in color.
• Verlagsort: Tokyo
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Botanik
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zoologie
• Themenwelt: Technik
• Schlagworte: CRISPR/Cas9 • Gene disruption • Gene Knockout • Genome editing • IPS • Knock-in • Talen • Targeted insertion • Transgenic Organisms • ZFN
• ISBN-10: 4-431-55227-8 / 4431552278
• ISBN-13: 978-4-431-55227-7 / 9784431552277