E-Book, Englisch, Band Volume 547, 506 Seiten
Reihe: Methods in Enzymology
E-Book, Englisch, Band Volume 547, 506 Seiten
Reihe: Methods in Enzymology
ISBN: 978-0-12-801615-2
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark
Autoren/Hrsg.
Weitere Infos & Material
1;Front Cover;1
2;The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations;4
3;Copyright;5
4;Contents;6
5;Contributors;14
6;Preface;20
7;Chapter One: In Vitro Enzymology of Cas9;22
7.1;1. Introduction;22
7.2;2. Expression and Purification of Cas9;24
7.2.1;Day 1: Cell transformation;25
7.2.2;Day 2: Culture growth and induction;25
7.2.3;Day 3: Cas9 purification by IMAC;26
7.2.4;Day 4: IEX and SEC chromatographic steps;27
7.2.5;Day 5: Concentration and storage;27
7.3;3. Preparation of Guide RNAs;28
7.3.1;Day 1: Preparation of transcription template;31
7.3.2;Day 2: In vitro transcription and gel purification;32
7.3.3;Day 3: Gel purification-continued;33
7.4;4. Endonuclease Cleavage Assays;34
7.4.1;Substrate preparation;35
7.4.2;Cleavage assay;36
7.4.3;Interpretation of cleavage assays;38
7.5;5. Concluding Remarks;38
7.6;Acknowledgments;38
7.7;References;39
8;Chapter Two: Targeted Genome Editing in Human Cells Using CRISPR/Cas Nucleases and Truncated Guide RNAs;42
8.1;1. Introduction;42
8.2;2. Methods;53
8.2.1;2.1. Identification of target sites using ZiFiT;53
8.2.1.1;Required materials;54
8.2.1.2;Ensure query sequence is valid;54
8.2.1.3;Design target sites;55
8.2.2;2.2. Construction of tru-gRNA expression plasmids;57
8.2.2.1;2.2.1. Reagents;57
8.2.2.2;2.2.2. Protocol;58
8.2.3;2.3. Transfection of sgRNA and Cas9 expression plasmids into human cells;59
8.2.3.1;2.3.1. Reagents;60
8.2.3.2;2.3.2. Protocol;60
8.2.3.2.1;2.3.2.1. Prior to Day 1;60
8.2.4;2.4. Quantitative T7EI assays to assess frequencies of targeted genome editing;61
8.2.4.1;2.4.1. Reagents;61
8.2.4.2;2.4.2. Protocol;63
8.3;Conflict of Interest;65
8.4;References;65
9;Chapter Three: Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes;68
9.1;1. Introduction;69
9.1.1;1.1. Introduction to programmable nucleases for genome editing;69
9.1.2;1.2. Overview of methods to study specificity of genome-editing agents;70
9.1.2.1;1.2.1. Discrete off-target site testing;70
9.1.2.2;1.2.2. Genome-wide selections;72
9.1.2.3;1.2.3. Minimally biased selections in vitro and in cells;74
9.1.3;1.3. Insights and improvements from ZFN specificity studies;78
9.1.4;1.4. Insights and improvements from TALEN specificity studies;80
9.1.5;1.5. Insights and improvements from Cas9 specificity studies;82
9.2;2. Methods;86
9.2.1;2.1. Overview of in vitro selection-based nuclease specificity profiling;86
9.2.2;2.2. Pre-selection library design;86
9.2.3;2.3. In vitro selection protocol;87
9.2.3.1;2.3.1. Before Day 1: Design and synthesize pre-selection library oligonucleotides;87
9.2.3.2;2.3.2. Day 1: Circularize library oligonucleotides;88
9.2.3.3;2.3.3. Day 2: Confirm circularization of library oligonucleotides and perform rolling-circle amplification;88
9.2.3.4;2.3.4. Day 3: Quantify and digest pre-selection library;88
9.2.3.5;2.3.5. Day 4: PCR of pre- and post-selection libraries;90
9.2.3.6;2.3.6. Day 5: High-throughput sequencing and analysis;91
9.2.4;2.4. Confirmation of in vitro-identified genomic off-target sites;92
9.3;3. Conclusion;94
9.4;Acknowledgments;94
9.5;References;95
10;Chapter Four: Genome Engineering with Custom Recombinases;100
10.1;1. Introduction;100
10.2;2. Target Identification;102
10.3;3. Recombinase Construction;103
10.4;4. Measurements of Recombinase Activity;106
10.4.1;4.1. Reporter plasmid construction;107
10.4.2;4.2. Luciferase assay;107
10.5;5. Site-Specific Integration;108
10.5.1;5.1. Donor plasmid construction;108
10.5.2;5.2. Cell culture methods;109
10.5.2.1;5.2.1. PCR confirmation of integration;109
10.5.2.2;5.2.2. Measurements of modification efficiency;110
10.5.2.3;5.2.3. Isolation and expansion of modified clones;110
10.6;6. Conclusions;111
10.7;Acknowledgments;111
10.8;References;111
11;Chapter Five: Genome Engineering in Human Cells;114
11.1;1. Introduction;115
11.2;2. Structure of the Human Genome;116
11.3;3. Scope of Human Gene Editing Using Programmable Nucleases;118
11.3.1;3.1. Gene disruption;118
11.3.2;3.2. Gene insertion;119
11.3.3;3.3. Gene correction;119
11.3.4;3.4. Chromosomal rearrangement;119
11.4;4. Programmable Nucleases Used for Genome Editing in Human Cells;120
11.4.1;4.1. ZFNs;120
11.4.2;4.2. TALENs;120
11.4.3;4.3. RGENs;123
11.5;5. Correction of Human Genetic Diseases Using Programmable Nucleases;124
11.6;6. Treatment of Human Nongenetic Diseases Using Programmable Nucleases;126
11.7;7. Genome Engineering in Human Pluripotent Stem Cells;127
11.8;8. Delivery of Programmable Nucleases to Human Cells;128
11.9;9. Nickases for Modifying the Human Genome;130
11.10;10. Enrichment of Gene-Edited Human Cells;131
11.11;11. Conclusion;132
11.12;Acknowledgments;132
11.13;References;133
12;Chapter Six: Genome Editing in Human Stem Cells;140
12.1;1. Introduction;141
12.2;2. Gene Targeting Strategies;142
12.3;3. Choice of Nuclease Targeting Sites;143
12.4;4. Experimental Procedures;144
12.4.1;4.1. Human iPSC culture and passaging;145
12.4.2;4.2. Preparation of plasmids for transient transfection;145
12.4.3;4.3. Nucleofection protocol;146
12.4.4;4.4. Verification of successful cutting and gene targeting;148
12.4.5;4.5. Cloning by single cell FACS sorting;149
12.4.6;4.6. Genotyping of clones;150
12.4.7;4.7. Verify iPSC pluripotency and quality;152
12.5;5. Alternative Approaches;152
12.5.1;5.1. Low transfection;152
12.5.2;5.2. Viral vectors;153
12.5.3;5.3. Off-targets;154
12.5.4;5.4. Cas9 nickases;155
12.5.5;5.5. Orthogonal Cas9 systems;156
12.6;References;156
13;Chapter Seven: Tagging Endogenous Loci for Live-Cell Fluorescence Imaging and Molecule Counting Using ZFNs, TALENs, and Cas9;160
13.1;1. Introduction;161
13.2;2. Methods;163
13.2.1;2.1. Donor plasmid design;163
13.2.1.1;2.1.1. Required materials;165
13.2.1.2;2.1.2. Option 1: Gibson assembly;165
13.2.1.3;2.1.3. Option 2: Classical cloning method;166
13.2.2;2.2. Generation of genome-edited cell lines using CRISPR, TALENs, or ZFNs;167
13.2.2.1;2.2.1. Required materials;167
13.2.2.2;2.2.2. Preparation of cells;168
13.2.2.3;2.2.3. Electroporation;170
13.2.2.4;2.2.4. Isolation of genome-edited cells;171
13.2.3;Genomic DNA extraction;173
13.2.4;PCR and sequencing;173
13.2.5;By immune blot:;174
13.2.6;By immunofluorescence microscopy;175
13.3;3. Tagging/Editing Limitations;175
13.4;4. Perspectives;177
13.4.1;4.1. Efficiency of cellular processes: Example of clathrin-mediated endocytosis;177
13.4.2;4.2. Quantification of protein stoichiometry in specific structures within genome-edited cells;177
13.4.3;4.3. Genome-edited stem cells: A new model for mammalian cell biology studies;178
13.5;Acknowledgments;179
13.6;References;179
14;Chapter Eight: Genome Editing Using Cas9 Nickases;182
14.1;1. Introduction;183
14.2;2. Target Selection;185
14.3;3. Plasmid sgRNA Construction;186
14.4;4. Validation of sgRNAs in Cell Lines;187
14.5;5. Cell Harvest and DNA Extraction;188
14.6;6. SURVEYOR Indel Analysis;189
14.7;7. HDR and Non-HDR Insertion Using Cas9n;191
14.8;8. Analysis of HDR and Insertion Events;192
14.9;9. Troubleshooting;193
14.10;Acknowledgments;194
14.11;References;194
15;Chapter Nine: Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and C...;196
15.1;1. Introduction;197
15.2;2. Cloning the Nickase and Catalytically Dead Variants of Cas9;198
15.2.1;2.1. The Cas9 endonuclease;198
15.2.2;2.2. Generating Cas9H840A and Cas9D10A/H840A expression vectors;200
15.2.3;2.3. Cloning and verifying the constructs;201
15.3;3. Selection of the Target Site and Cloning of sgRNA Constructs;202
15.3.1;3.1. Selecting suitable target sequences;202
15.3.2;3.2. Cloning the guide RNA constructs;203
15.4;4. Cell Transfection and FACS Analysis;204
15.4.1;4.1. Transfection;206
15.4.2;4.2. Analysis and interpretation of the results;209
15.5;5. Materials;210
15.5.1;5.1. Cloning;210
15.5.2;5.2. Cell culture, transfections, data collection, and analysis;210
15.6;6. Summary;211
15.7;References;211
16;Chapter Ten: Adapting CRISPR/Cas9 for Functional Genomics Screens;214
16.1;1. Introduction;215
16.2;2. Altering the Vector Design for High-Throughput Screens;216
16.3;3. Construction of sgRNA Libraries;220
16.3.1;3.1. Guide sequence prediction;220
16.3.2;3.2. Cloning of guide templates;223
16.3.2.1;3.2.1. Layout of the guide template;223
16.3.2.2;3.2.2. Initial guide library preparation;224
16.3.2.3;3.2.3. PCR amplification of pooled oligonucleotide templates;224
16.3.2.3.1;Reagent amounts;224
16.3.2.3.2;Thermocycler reaction conditions;224
16.3.2.3.3;Reagent amounts;224
16.3.2.3.4;Thermocycler reaction conditions;225
16.3.2.4;3.2.4. Digestion and ligation of the guides into vector backbone;225
16.3.2.5;3.2.5. Assessing ligation efficiency;225
16.3.2.6;3.2.6. Large-scale transformation of the guide library;225
16.3.2.7;3.2.7. Checking the quality of the guide library;226
16.3.2.8;3.2.8. Bulk harvesting of bacterial-transformed guide library;226
16.3.2.9;3.2.9. Arraying individual bacterial guide library clones;226
16.4;4. Retroviral Transduction of the Guide Library;227
16.5;5. Notes on Screening Design Parameters;228
16.6;6. Decoding ``Hits´´ from Positive Selection Screens Involving sgRNA Library Pools;231
16.6.1;Reagent amounts;231
16.6.2;Thermocycler reaction conditions;231
16.7;7. Conclusion;232
16.8;References;232
17;Chapter Eleven: The iCRISPR Platform for Rapid Genome Editing in Human Pluripotent Stem Cells;236
17.1;1. Introduction;237
17.2;2. Generation of iCas9 hPSCs;241
17.2.1;2.1. Vector design;243
17.2.1.1;2.1.1. TALEN vectors;243
17.2.1.2;2.1.2. Donor vectors;243
17.2.2;2.2. hPSC electroporation;243
17.2.3;2.3. Selection and expansion of clonal lines;245
17.2.4;2.4. Genotyping by Southern blot;246
17.2.5;2.5. Validation;250
17.2.5.1;2.5.1. RT-PCR analysis;250
17.2.5.2;2.5.2. Immunohistochemical analysis of pluripotency marker expression;251
17.2.5.3;2.5.3. Teratoma assay;251
17.3;3. Generation of Knockout hPSCs Using iCRISPR;252
17.3.1;3.1. sgRNA design;252
17.3.2;3.2. sgRNA production;252
17.3.2.1;3.2.1. PCR amplification of in vitro transcription (IVT) DNA templates;252
17.3.2.2;3.2.2. In vitro transcription and purification of sgRNAs;254
17.3.3;3.3. Single or multiplex sgRNA transfection in hPSCs;254
17.3.4;3.4. Assessment of Indel frequency;255
17.3.4.1;3.4.1. PCR amplification of the CRISPR target region;255
17.3.4.2;3.4.2. Quantification of Indels through T7EI assay;256
17.3.4.2.1;3.4.2.1. Hybridization;256
17.3.4.2.2;3.4.2.2. Digestion;256
17.3.4.2.3;3.4.2.3. Quantification;257
17.3.4.3;3.4.3. Quantification of Indels through RFLP assay;257
17.3.4.3.1;3.4.3.1. Digestion;257
17.3.4.3.2;3.4.3.2. Quantification;257
17.3.5;3.5. Clonal expansion of knockout lines;258
17.3.5.1;3.5.1. Replating and colony picking;258
17.3.5.2;3.5.2. Colony screening;258
17.3.5.2.1;3.5.2.1. Lysis;259
17.3.5.2.2;3.5.2.2. PCR and sequencing;259
17.3.5.3;3.5.3. Validation;260
17.3.5.3.1;3.5.3.1. Validation of the mutant alleles;260
17.3.5.3.2;3.5.3.2. Off-target analysis;260
17.4;4. Generation of Precise Nucleotide Alterations Using iCRISPR;260
17.4.1;4.1. Design of ssDNA as HDR templates;261
17.4.2;4.2. ssDNA/sgRNA cotransfection in hPSCs;262
17.4.3;4.3. Establishment of clonal lines;262
17.5;5. Inducible Gene Knockout in hPSCs Using iCRISPR;263
17.5.1;5.1. Inducible gene knockout through sgRNA transfection;264
17.5.2;5.2. Inducible gene knockout through using iCr hPSC lines;264
17.6;6. Conclusions and Future Directions;265
17.6.1;6.1. Anticipated results;265
17.6.2;6.2. In-frame mutations;265
17.6.3;6.3. Cross contamination;266
17.6.4;6.4. Time and throughput considerations;266
17.6.5;6.5. Off-target considerations;266
17.6.6;6.6. Additional use and extension of the iCRISPR platform;267
17.7;Acknowledgments;268
17.8;References;268
18;Chapter Twelve: Creating Cancer Translocations in Human Cells Using Cas9 DSBs and nCas9 Paired Nicks;272
18.1;1. Introduction;273
18.2;2. Materials;275
18.2.1;2.1. Cas9, nCas9, and sgRNA expression plasmid preparation;275
18.2.2;2.2. Cell culture and transfection;275
18.2.3;2.3. T7 endonuclease I assay;276
18.2.4;2.4. PCR detection of translocations;276
18.2.5;2.5. PCR quantification of translocations;276
18.3;3. Methods to Induce and Detect Cancer Translocations in Human Cells;277
18.3.1;3.1. sgRNA design and expression plasmid construction;277
18.3.2;3.2. Cell transfections with sgRNA and Cas9 or nCas9 expression plasmids;281
18.3.3;3.3. T7 endonuclease I assay to estimate cleavage efficiency;283
18.3.4;3.4. PCR-based translocation detection;284
18.3.5;3.5. Quantification of potential off-target cleavage;285
18.3.6;3.6. Quantification of translocation frequency using a 96-well plate screen;287
18.3.7;3.7. Translocation frequency determination by serial dilution;289
18.4;4. Conclusions;290
18.5;Acknowledgments;290
18.6;References;290
19;Chapter Thirteen: Genome Editing for Human Gene Therapy;294
19.1;1. Introduction;295
19.2;2. Genome Editing of B2M in Primary Human CD4+ T Cells;297
19.2.1;2.1. Required materials;298
19.2.2;2.2. Isolation of CD4+ T cells from peripheral blood;299
19.2.2.1;Notes;300
19.2.3;2.3. Delivery of CRISPR/Cas9 by nucleofection;300
19.2.3.1;2.3.1. Nucleofection;301
19.2.3.2;2.3.2. Postnucleofection;303
19.2.3.2.1;Notes;303
19.2.4;2.4. Evaluation of targeting efficiency;303
19.2.4.1;2.4.1. FACS-based analysis;304
19.2.4.2;2.4.2. PCR-based screening assay;305
19.2.4.2.1;Notes;306
19.3;3. Targeting of CCR5 in Human CD34+ HSPCs Using CRISPR/Cas9;307
19.3.1;3.1. Required materials;309
19.3.2;3.2. Transfection of CD34+ HSPCs;310
19.3.2.1;3.2.1. Isolation of CD34+ HSPCs from cord blood;310
19.3.2.2;3.2.2. Nucleofection of CD34+ HSPCs;311
19.3.2.3;3.2.3. Cell sorting;311
19.3.2.3.1;Notes;312
19.3.3;3.3. Colony-forming cell assay;312
19.3.3.1;Notes;313
19.3.4;3.4. Clonal analysis;313
19.3.4.1;Notes;314
19.4;References;314
20;Chapter Fourteen: Generation of Site-Specific Mutations in the Rat Genome Via CRISPR/Cas9;318
20.1;1. Theory;319
20.2;2. Equipment;321
20.3;3. Materials;322
20.3.1;3.1. Solutions and buffers;323
20.4;4. Protocol;324
20.4.1;4.1. Preparation;324
20.4.2;4.2. Duration;324
20.4.3;4.3. Caution;325
20.5;5. Step 1: In Vitro Transcription of sgRNA Target Oligonucleotides;325
20.5.1;5.1. Overview;325
20.5.2;5.2. Duration;325
20.5.3;5.3. Tip;326
20.6;6. Step 2: In Vitro Transcription of Cas9 mRNA;328
20.6.1;6.1. Overview;328
20.6.2;6.2. Duration;328
20.6.3;6.3. Tip;329
20.7;7. Step 3: Preparation of Pseudopregnant Female Rats and One-Cell Rat Embryos;330
20.7.1;7.1. Overview;330
20.7.2;7.2. Duration;330
20.7.3;7.3. Tip;330
20.7.4;7.4. Tip;331
20.7.5;7.5. Tip;331
20.8;8. Step 4: Microinjection of One-Cell Embryos and Transplanting the Embryos into Pseudopregnant Rats;332
20.8.1;8.1. Overview;332
20.8.2;8.2. Duration;332
20.8.3;8.3. Tip;333
20.8.4;8.4. Tip;333
20.8.5;8.5. Tip;333
20.8.6;8.6. Tip;333
20.8.7;8.7. Tip;333
20.8.8;8.8. Tip;335
20.9;9. Step 5: Identification of Founder Rats;335
20.9.1;9.1. Overview;335
20.9.2;9.2. Duration;335
20.9.3;9.3. Tip;336
20.9.4;9.4. Tip;336
20.10;10. Step 6: Production of F1 Generation Rats;338
20.10.1;10.1. Overview;338
20.10.2;10.2. Duration;338
20.10.3;10.3. Tips;338
20.11;References;338
21;Chapter Fifteen: CRISPR/Cas9-Based Genome Editing in Mice by Single Plasmid Injection;340
21.1;1. Introduction;341
21.2;2. Design and Construction of CRISPR/Cas9 Plasmids with pX330;343
21.2.1;2.1. Selection and off-target analysis of sgRNA in targeted gene;343
21.2.1.1;2.1.1. Design of sgRNAs against the target gene: Protocol;344
21.2.2;2.2. Construction of pX330 with designed sgRNA;344
21.2.2.1;2.2.1. Insertion of sgRNA into the pX330 plasmid: Protocol;344
21.3;3. Validation of pX330 In Vitro;347
21.3.1;3.1. Construction of pCAG-EGxxFP with the targeted genomic region;347
21.3.1.1;3.1.1. Insertion of the target genomic fragment into pCAG-EGxxFP plasmid: Protocol;348
21.3.2;3.2. Cotransfection of pX330-sgRNA and pCAG-EGxxFP-target into HEK293T cells;349
21.3.2.1;3.2.1. Cell culture and transfection in HEK293T cells: Protocol;349
21.3.3;3.3. Observation of EGFP fluorescence in the transfected cells;350
21.4;4. One-Step Generation of Mutant Mice Via Circular Plasmid Injection;351
21.4.1;4.1. Collecting the fertilized eggs;351
21.4.1.1;4.1.1. Superovulation treatment and collection of fertilized eggs: Protocol;351
21.4.2;4.2. Preparing pX330-sgRNA plasmid for microinjection;351
21.4.2.1;4.2.1. Preparation of pX330-sgRNA plasmid for microinjection: Protocol;352
21.4.3;4.3. Pronuclear microinjection of circular pX330-sgRNA plasmid;352
21.4.3.1;4.3.1. Manipulating mouse embryos and microinjection system: Protocol;352
21.5;5. Screening for Targeted Mutation in Mice;353
21.5.1;5.1. Direct sequencing of PCR products: Protocol;353
21.6;6. Concluding Remarks;353
21.7;Acknowledgment;356
21.8;References;356
22;Chapter Sixteen: Imaging Genomic Elements in Living Cells Using CRISPR/Cas9;358
22.1;1. Introduction;359
22.1.1;1.1. Choice of target sites and DNA recognition methods;359
22.1.2;1.2. Sensitivity and specificity of genome imaging using CRISPR/Cas9;361
22.2;2. Generation of Cell Lines Stably Expressing dCas9-GFP;362
22.2.1;2.1. Generation of dCas9-GFP constructs;362
22.2.2;2.2. dCas9-GFP/Tet-On 3G lentiviral production;363
22.2.3;2.3. dCas9-GFP/Tet-On 3G lentiviral infection;364
22.2.4;2.4. Selection of clonal cell lines stably expressing dCas9-GFP;366
22.3;3. Expression of sgRNAs Using Lentiviral Vector;367
22.3.1;3.1. sgRNA design and cloning;367
22.3.2;3.2. sgRNA lentiviral infection;368
22.4;4. Labeling of Nonrepetitive Sequences;368
22.4.1;4.1. Target selection and sgRNA design;368
22.4.2;4.2. High-throughput sgRNA cloning;369
22.4.3;4.3. Production of pooled sgRNA lentiviruses;370
22.5;5. Imaging of Genomic Loci Detected by CRISPR;370
22.5.1;5.1. Verify CRISPR signal by a modified FISH staining protocol;370
22.5.2;5.2. Live-cell imaging of genomic loci;371
22.6;6. Summary;373
22.7;Acknowledgments;374
22.8;References;374
23;Chapter Seventeen: Cas9-Based Genome Editing in Xenopus tropicalis;376
23.1;1. Introduction;377
23.2;2. Principle;378
23.3;3. Protocol;380
23.3.1;3.1. Background knowledge and experimental equipment;380
23.3.2;3.2. sgRNA design;380
23.3.2.1;3.2.1. Considerations in target site choice;382
23.3.3;3.3. sgRNA template construction;383
23.3.3.1;3.3.1. Template assembly by PCR: Primers;383
23.3.3.2;3.3.2. Template assembly by PCR: Assembly conditions;383
23.3.3.3;3.3.3. In vitro transcription of sgRNA;384
23.3.4;3.4. Procedure for microinjection;385
23.3.4.1;3.4.1. Doses of sgRNA and Cas9;385
23.3.4.2;3.4.2. Sidebar: Cas9 protein in vitro cleavage assays;388
23.3.4.3;3.4.3. Procedure for embryo microinjection;388
23.3.5;3.5. Assessment of mutagenesis: Genotyping;389
23.3.5.1;3.5.1. Embryo lysis and PCR;389
23.3.5.2;3.5.2. Evaluation of sequencing results and subsequent identification of specific indels;390
23.4;4. Discussion;391
23.4.1;4.1. Multiple targeting strategy: Avoiding off-target problems and simpler genotyping of F1 animals;391
23.4.2;4.2. Further applications of CRISPR-mediated mutagenesis in Xenopus;393
23.5;Acknowledgments;394
23.6;References;394
24;Chapter Eighteen: Cas9-Based Genome Editing in Zebrafish;398
24.1;1. Introduction;399
24.1.1;1.1. CRISPR/Cas adaptive immunity;399
24.1.2;1.2. The Type II CRISPR/Cas system;400
24.1.3;1.3. The development of CRISPR/Cas genome-editing technology;401
24.1.4;1.4. The zebrafish animal model and CRISPR/Cas;404
24.2;2. Targeted Generation of Indel Mutations;406
24.2.1;2.1. Cas9 modification and delivery platforms;406
24.2.1.1;Protocol for preparation of SpCas9 mRNA for microinjection;407
24.2.2;2.2. Single-guide RNA design considerations;409
24.2.2.1;Protocol for preparation of sgRNAs for microinjection:;413
24.2.3;2.3. Introduction and identification of Cas9-sgRNA-induced indels;416
24.3;3. Other Targeted Genome-Editing Strategies;417
24.3.1;3.1. Precise sequence modifications mediated by single-stranded oligonucleotides;417
24.3.2;3.2. Targeted integration of long DNA fragments;418
24.3.3;3.3. Chromosomal deletions and other rearrangements;421
24.4;4. Future Directions;422
24.5;Acknowledgments;424
24.6;References;424
25;Chapter Nineteen: Cas9-Based Genome Editing in Drosophila;436
25.1;1. Introduction;436
25.2;2. Applications and Design Considerations for CRISPR-Based Genome Editing;438
25.2.1;2.1. Selection of sgRNA target sites;440
25.2.2;2.2. Tools facilitating sgRNA design;441
25.3;3. Delivery of CRISPR Components;442
25.4;4. Generation of CRISPR Reagents;444
25.4.1;4.1. Cloning of sgRNAs into expression vectors;445
25.4.1.1;Materials;446
25.4.1.2;Protocol;446
25.4.2;4.2. Cloning of donor constructs;447
25.4.2.1;Materials;448
25.4.2.2;Protocol;449
25.4.3;4.3. Isolation of in vivo genome modifications;450
25.5;5. Detection of Mutations;450
25.5.1;5.1. Preparation of genomic DNA from fly wings;451
25.5.1.1;5.1.1. Restriction profiling;452
25.5.1.1.1;Materials;452
25.5.1.1.2;Protocol;452
25.5.1.2;5.1.2. Surveyor assay to detect indels;453
25.5.1.2.1;Materials;454
25.5.1.2.2;Protocol;454
25.5.1.3;5.1.3. Detection of mutations using HRMA;455
25.5.1.3.1;Materials;456
25.5.1.3.2;Protocol;456
25.5.2;5.2. Analysis of HRMA data;457
25.6;Acknowledgments;457
25.7;References;458
26;Chapter Twenty: Transgene-Free Genome Editing by Germline Injection of CRISPR/Cas RNA;462
26.1;1. Theory, Philosophy, and Practical Considerations;463
26.1.1;1.1. Overview;463
26.1.2;1.2. When to use or not to use transgenes for delivery of CRISPR/Cas;464
26.1.3;1.3. Altered mutation profile from transgene-free treatment with CRISPR/Cas;465
26.1.4;1.4. A note on specificity of CRISPR/Cas cleavage;467
26.2;2. Equipment;467
26.3;3. Materials;468
26.4;4. Identifying a Target Sequence;468
26.5;5. Generating Your sgRNA Construct;470
26.5.1;5.1. Oligonucleotide design;470
26.5.2;5.2. Insert generation;470
26.5.3;5.3. Preparation of linearized vector for the sgRNA construct;471
26.5.4;5.4. Construction and identification of sgRNA synthesis plasmid;471
26.6;6. In Vitro Synthesis of sgRNA;472
26.6.1;6.1. Linearization of sgRNA template plasmid;472
26.6.2;6.2. In vitro transcription to generate sgRNA;473
26.6.3;6.3. Purification of in vitro-transcribed sgRNA;473
26.7;7. In Vitro Synthesis of hCas9 mRNA;473
26.7.1;7.1. Linearization of SP6-hCas9-Ce-mRNA plasmid;473
26.7.2;7.2. In vitro transcription of hCas9 mRNA;474
26.7.3;7.3. Polyadenylation of in vitro-transcribed hCas9 mRNA;474
26.7.4;7.4. Purification of in vitro-transcribed, polyadenylated hCas9 mRNA;474
26.8;8. Injection of sgRNA and mRNA;474
26.9;9. Recovery of Mutants Generated Using CRISPR/Cas;475
26.9.1;9.1. Recovery and plating of injected animals;475
26.9.2;9.2. Identification of animals carrying mutations induced by CRISPR/Cas;476
26.10;References;476
27;Chapter Twenty-One: Cas9-Based Genome Editing in Arabidopsis and Tobacco;480
27.1;1. Introduction;481
27.2;2. Cas9 and sgRNA expression;482
27.3;3. Dual sgRNA-Guided Genome Editing;484
27.3.1;3.1. Designing and constructing dual sgRNAs;484
27.3.2;3.2. Transfecting and expressing Cas9/sgRNAs in protoplasts;485
27.3.3;3.3. Evaluating the frequency of targeted genome modifications;486
27.4;4. Perspectives;488
27.5;5. Notes;489
27.6;Acknowledgments;491
27.7;References;491
28;Chapter Twenty-Two: Multiplex Engineering of Industrial Yeast Genomes Using CRISPRm;494
28.1;1. Introduction;495
28.2;2. Plasmid Design;497
28.3;3. Cas9 Expression;499
28.4;4. Guide RNA Expression;499
28.5;5. Screening Method;502
28.5.1;5.1. Cloning the target sequence into pCAS;503
28.5.2;5.2. Double-stranded linear DNA repair oligos;503
28.5.3;5.3. CRISPRm screening consists of the cotransformation of pCAS and the double-stranded linear DNA homologous repair template;504
28.5.4;5.4. Industrial yeast;506
28.5.5;5.5. Markerless gene assembly in the yeast chromosome;506
28.6;6. Concluding Remarks;508
28.7;Acknowledgments;509
28.8;References;509
29;Chapter Twenty-Three: Protein Engineering of Cas9 for Enhanced Function;512
29.1;1. Introduction;513
29.1.1;1.1. The structure of Cas9;515
29.1.2;1.2. Current uses;518
29.1.3;1.3. Initial engineering questions;518
29.2;2. Methods;519
29.2.1;2.1. A note on applications;519
29.2.2;2.2. Electrocompetent E. coli preparation for library construction;520
29.2.3;2.3. Discovery of functional, engineered, variants of Cas9 proteins;521
29.2.4;2.4. Screening Cas9;521
29.2.5;2.5. Selecting Cas9;521
29.2.6;2.6. Screening for functional Cas9 variants;523
29.2.7;2.7. Determining screening enrichment of PDZ-dCas9 domain insertions;525
29.2.8;2.8. Identifying and testing PDZ-Cas9 clones from a screened library;527
29.2.9;2.9. Expanding horizons;528
29.3;3. Conclusion;529
29.4;References;529
30;Author Index;534
31;Subject Index;560
32;Color Plate;572
Contributors
Hossein Aleyasin, Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, and Fishberg Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA Ishraq Alim, Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA A. Ambrus, Department of Medical Biochemistry, Semmelweis University, and MTA-SE Laboratory for Neurobiochemistry, Budapest, Hungary Estela Area-Gomez, Department of Neurology, Columbia University Medical Center, New York, USA Sandra R. Bacman, Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Stephen D. Baird, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada Irene Bolea, Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA David C. Chan, Division of Biology and Biological Engineering, and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA Guo Chen, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China Linbo Chen, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China Quan Chen, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, and State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Andy Cheuk-Him Ng, Children’s Hospital of Eastern Ontario Research Institute, and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada Megan M. Cleland, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA Swathi Devireddy, Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Ajit S. Divakaruni, Department of Pharmacology, University of California, San Diego, California, USA Du Feng, Guangdong Key laboratory of Age-related Cardiac-cerebral Vascular Disease, Institute of Neurology, Guangdong Medical College, Zhanjiang, Guangdong Province, China David A. Ferrick, Seahorse Bioscience, Billerica, Massachusetts, USA Wen-Biao Gan, Department of Physiology and Neuroscience, Skirball Institute, New York University School of Medicine, New York, USA Elisabeth Garland-Kuntz, Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Shealinna X. Ge, Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, Maryland, USA Roberta A. Gottlieb, Department of Molecular Cardiobiology, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA Hengchang Guo, Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, and Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA Renee E. Haskew-Layton, Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, and Department of Health and Natural Sciences, Mercy College, Dobbs Ferry, New York, USA Riikka H. Hämäläinen, Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland Peter J. Hollenbeck, Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Gregory P. Holmes-Hampton, Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA Martin Jastroch, Institute for Diabetes and Obesity, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Mariusz Karbowski, Center for Biomedical Engineering and Technology, and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA Adam L. Knight, Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Pin-Chao Liao, Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA Mei-Yao Lin, Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA Lei Liu, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Jordi Magrané, Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA Giovanni Manfedi, Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, USA Thomas Misgeld, German Center for Neurodegenerative Diseases (DZNE); Munich Center for Systems Neurology (SyNergy), and Institute of Neuronal Cell Biology, Technische Universität München, Munich, Germany Carlos T. Moraes, Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Anne N. Murphy, Department of Pharmacology, University of California, San Diego, California, USA David G. Nicholls, Department of Clinical Sciences in Malmö, Unit of Molecular Metabolism, Lund University Diabetes Centre, CRC, Malmö, Sweden, and Buck Institute for Research on Aging, Novato, California, USA Dominik Paquet, Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, and German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Alexander Paradyse, Department of Pharmacology, University of California, San Diego, California, USA Guy A. Perkins, National Center for Microscopy and Imaging Research, University of California, San Diego, California, USA Anh H. Pham, Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA Milena Pinto, Department of Neurology, University of Miami School of Medicine, Miami, Florida, USA Gabriela Plucinska, Munich Center for Systems Neurology (SyNergy), Munich, Germany Brian M. Polster, Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR), and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA Rajiv R. Ratan, Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA Brian A. Roelofs, Center for Biomedical Engineering and Technology, and Department of Biochemistry and Molecular Biology,...
