This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers recent research and methods development for changing the DNA sequence within the genomes of cells and organisms. Focusing on enzymes that generate double-strand breaks in DNA, the chapters describe use of molecular tools to introduce or delete genetic information at specific sites in the genomes of animal, plant and bacterial cells. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field- Covers research methods in biomineralization science- Contains sections on such topics as genome editing, genome engineering, CRISPR, Cas9, TALEN and zinc finger nuclease
Front Cover 1
The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations 4
Copyright 5
Contents 6
Contributors 14
Preface 20
Chapter One: In Vitro Enzymology of Cas9 22
1. Introduction 22
2. Expression and Purification of Cas9 24
Day 1: Cell transformation 25
Day 2: Culture growth and induction 25
Day 3: Cas9 purification by IMAC 26
Day 4: IEX and SEC chromatographic steps 27
Day 5: Concentration and storage 27
3. Preparation of Guide RNAs 28
Day 1: Preparation of transcription template 31
Day 2: In vitro transcription and gel purification 32
Day 3: Gel purification-continued 33
4. Endonuclease Cleavage Assays 34
Substrate preparation 35
Cleavage assay 36
Interpretation of cleavage assays 38
5. Concluding Remarks 38
Acknowledgments 38
References 39
Chapter Two: Targeted Genome Editing in Human Cells Using CRISPR/Cas Nucleases and Truncated Guide RNAs 42
1. Introduction 42
2. Methods 53
2.1. Identification of target sites using ZiFiT 53
Required materials 54
Ensure query sequence is valid 54
Design target sites 55
2.2. Construction of tru-gRNA expression plasmids 57
2.2.1. Reagents 57
2.2.2. Protocol 58
2.3. Transfection of sgRNA and Cas9 expression plasmids into human cells 59
2.3.1. Reagents 60
2.3.2. Protocol 60
2.3.2.1. Prior to Day 1 60
2.4. Quantitative T7EI assays to assess frequencies of targeted genome editing 61
2.4.1. Reagents 61
2.4.2. Protocol 63
Conflict of Interest 65
References 65
Chapter Three: Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes 68
1. Introduction 69
1.1. Introduction to programmable nucleases for genome editing 69
1.2. Overview of methods to study specificity of genome-editing agents 70
1.2.1. Discrete off-target site testing 70
1.2.2. Genome-wide selections 72
1.2.3. Minimally biased selections in vitro and in cells 74
1.3. Insights and improvements from ZFN specificity studies 78
1.4. Insights and improvements from TALEN specificity studies 80
1.5. Insights and improvements from Cas9 specificity studies 82
2. Methods 86
2.1. Overview of in vitro selection-based nuclease specificity profiling 86
2.2. Pre-selection library design 86
2.3. In vitro selection protocol 87
2.3.1. Before Day 1: Design and synthesize pre-selection library oligonucleotides 87
2.3.2. Day 1: Circularize library oligonucleotides 88
2.3.3. Day 2: Confirm circularization of library oligonucleotides and perform rolling-circle amplification 88
2.3.4. Day 3: Quantify and digest pre-selection library 88
2.3.5. Day 4: PCR of pre- and post-selection libraries 90
2.3.6. Day 5: High-throughput sequencing and analysis 91
2.4. Confirmation of in vitro-identified genomic off-target sites 92
3. Conclusion 94
Acknowledgments 94
References 95
Chapter Four: Genome Engineering with Custom Recombinases 100
1. Introduction 100
2. Target Identification 102
3. Recombinase Construction 103
4. Measurements of Recombinase Activity 106
4.1. Reporter plasmid construction 107
4.2. Luciferase assay 107
5. Site-Specific Integration 108
5.1. Donor plasmid construction 108
5.2. Cell culture methods 109
5.2.1. PCR confirmation of integration 109
5.2.2. Measurements of modification efficiency 110
5.2.3. Isolation and expansion of modified clones 110
6. Conclusions 111
Acknowledgments 111
References 111
Chapter Five: Genome Engineering in Human Cells 114
1. Introduction 115
2. Structure of the Human Genome 116
3. Scope of Human Gene Editing Using Programmable Nucleases 118
3.1. Gene disruption 118
3.2. Gene insertion 119
3.3. Gene correction 119
3.4. Chromosomal rearrangement 119
4. Programmable Nucleases Used for Genome Editing in Human Cells 120
4.1. ZFNs 120
4.2. TALENs 120
4.3. RGENs 123
5. Correction of Human Genetic Diseases Using Programmable Nucleases 124
6. Treatment of Human Nongenetic Diseases Using Programmable Nucleases 126
7. Genome Engineering in Human Pluripotent Stem Cells 127
8. Delivery of Programmable Nucleases to Human Cells 128
9. Nickases for Modifying the Human Genome 130
10. Enrichment of Gene-Edited Human Cells 131
11. Conclusion 132
Acknowledgments 132
References 133
Chapter Six: Genome Editing in Human Stem Cells 140
1. Introduction 141
2. Gene Targeting Strategies 142
3. Choice of Nuclease Targeting Sites 143
4. Experimental Procedures 144
4.1. Human iPSC culture and passaging 145
4.2. Preparation of plasmids for transient transfection 145
4.3. Nucleofection protocol 146
4.4. Verification of successful cutting and gene targeting 148
4.5. Cloning by single cell FACS sorting 149
4.6. Genotyping of clones 150
4.7. Verify iPSC pluripotency and quality 152
5. Alternative Approaches 152
5.1. Low transfection 152
5.2. Viral vectors 153
5.3. Off-targets 154
5.4. Cas9 nickases 155
5.5. Orthogonal Cas9 systems 156
References 156
Chapter Seven: Tagging Endogenous Loci for Live-Cell Fluorescence Imaging and Molecule Counting Using ZFNs, TALENs, and Cas9 160
1. Introduction 161
2. Methods 163
2.1. Donor plasmid design 163
2.1.1. Required materials 165
2.1.2. Option 1: Gibson assembly 165
2.1.3. Option 2: Classical cloning method 166
2.2. Generation of genome-edited cell lines using CRISPR, TALENs, or ZFNs 167
2.2.1. Required materials 167
2.2.2. Preparation of cells 168
2.2.3. Electroporation 170
2.2.4. Isolation of genome-edited cells 171
Genomic DNA extraction 173
PCR and sequencing 173
By immune blot: 174
By immunofluorescence microscopy 175
3. Tagging/Editing Limitations 175
4. Perspectives 177
4.1. Efficiency of cellular processes: Example of clathrin-mediated endocytosis 177
4.2. Quantification of protein stoichiometry in specific structures within genome-edited cells 177
4.3. Genome-edited stem cells: A new model for mammalian cell biology studies 178
Acknowledgments 179
References 179
Chapter Eight: Genome Editing Using Cas9 Nickases 182
1. Introduction 183
2. Target Selection 185
3. Plasmid sgRNA Construction 186
4. Validation of sgRNAs in Cell Lines 187
5. Cell Harvest and DNA Extraction 188
6. SURVEYOR Indel Analysis 189
7. HDR and Non-HDR Insertion Using Cas9n 191
8. Analysis of HDR and Insertion Events 192
9. Troubleshooting 193
Acknowledgments 194
References 194
Chapter Nine: Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and C... 196
1. Introduction 197
2. Cloning the Nickase and Catalytically Dead Variants of Cas9 198
2.1. The Cas9 endonuclease 198
2.2. Generating Cas9H840A and Cas9D10A/H840A expression vectors 200
2.3. Cloning and verifying the constructs 201
3. Selection of the Target Site and Cloning of sgRNA Constructs 202
3.1. Selecting suitable target sequences 202
3.2. Cloning the guide RNA constructs 203
4. Cell Transfection and FACS Analysis 204
4.1. Transfection 206
4.2. Analysis and interpretation of the results 209
5. Materials 210
5.1. Cloning 210
5.2. Cell culture, transfections, data collection, and analysis 210
6. Summary 211
References 211
Chapter Ten: Adapting CRISPR/Cas9 for Functional Genomics Screens 214
1. Introduction 215
2. Altering the Vector Design for High-Throughput Screens 216
3. Construction of sgRNA Libraries 220
3.1. Guide sequence prediction 220
3.2. Cloning of guide templates 223
3.2.1. Layout of the guide template 223
3.2.2. Initial guide library preparation 224
3.2.3. PCR amplification of pooled oligonucleotide templates 224
Reagent amounts 224
Thermocycler reaction conditions 224
Reagent amounts 224
Thermocycler reaction conditions 225
3.2.4. Digestion and ligation of the guides into vector backbone 225
3.2.5. Assessing ligation efficiency 225
3.2.6. Large-scale transformation of the guide library 225
3.2.7. Checking the quality of the guide library 226
3.2.8. Bulk harvesting of bacterial-transformed guide library 226
3.2.9. Arraying individual bacterial guide library clones 226
4. Retroviral Transduction of the Guide Library 227
5. Notes on Screening Design Parameters 228
6. Decoding ``Hits´´ from Positive Selection Screens Involving sgRNA Library Pools 231
Reagent amounts 231
Thermocycler reaction conditions 231
7. Conclusion 232
References 232
Chapter Eleven: The iCRISPR Platform for Rapid Genome Editing in Human Pluripotent Stem Cells 236
1. Introduction 237
2. Generation of iCas9 hPSCs 241
2.1. Vector design 243
2.1.1. TALEN vectors 243
2.1.2. Donor vectors 243
2.2. hPSC electroporation 243
2.3. Selection and expansion of clonal lines 245
2.4. Genotyping by Southern blot 246
2.5. Validation 250
2.5.1. RT-PCR analysis 250
2.5.2. Immunohistochemical analysis of pluripotency marker expression 251
2.5.3. Teratoma assay 251
3. Generation of Knockout hPSCs Using iCRISPR 252
3.1. sgRNA design 252
3.2. sgRNA production 252
3.2.1. PCR amplification of in vitro transcription (IVT) DNA templates 252
3.2.2. In vitro transcription and purification of sgRNAs 254
3.3. Single or multiplex sgRNA transfection in hPSCs 254
3.4. Assessment of Indel frequency 255
3.4.1. PCR amplification of the CRISPR target region 255
3.4.2. Quantification of Indels through T7EI assay 256
3.4.2.1. Hybridization 256
3.4.2.2. Digestion 256
3.4.2.3. Quantification 257
3.4.3. Quantification of Indels through RFLP assay 257
3.4.3.1. Digestion 257
3.4.3.2. Quantification 257
3.5. Clonal expansion of knockout lines 258
3.5.1. Replating and colony picking 258
3.5.2. Colony screening 258
3.5.2.1. Lysis 259
3.5.2.2. PCR and sequencing 259
3.5.3. Validation 260
3.5.3.1. Validation of the mutant alleles 260
3.5.3.2. Off-target analysis 260
4. Generation of Precise Nucleotide Alterations Using iCRISPR 260
4.1. Design of ssDNA as HDR templates 261
4.2. ssDNA/sgRNA cotransfection in hPSCs 262
4.3. Establishment of clonal lines 262
5. Inducible Gene Knockout in hPSCs Using iCRISPR 263
5.1. Inducible gene knockout through sgRNA transfection 264
5.2. Inducible gene knockout through using iCr hPSC lines 264
6. Conclusions and Future Directions 265
6.1. Anticipated results 265
6.2. In-frame mutations 265
6.3. Cross contamination 266
6.4. Time and throughput considerations 266
6.5. Off-target considerations 266
6.6. Additional use and extension of the iCRISPR platform 267
Acknowledgments 268
References 268
Chapter Twelve: Creating Cancer Translocations in Human Cells Using Cas9 DSBs and nCas9 Paired Nicks 272
1. Introduction 273
2. Materials 275
2.1. Cas9, nCas9, and sgRNA expression plasmid preparation 275
2.2. Cell culture and transfection 275
2.3. T7 endonuclease I assay 276
2.4. PCR detection of translocations 276
2.5. PCR quantification of translocations 276
3. Methods to Induce and Detect Cancer Translocations in Human Cells 277
3.1. sgRNA design and expression plasmid construction 277
3.2. Cell transfections with sgRNA and Cas9 or nCas9 expression plasmids 281
3.3. T7 endonuclease I assay to estimate cleavage efficiency 283
3.4. PCR-based translocation detection 284
3.5. Quantification of potential off-target cleavage 285
3.6. Quantification of translocation frequency using a 96-well plate screen 287
3.7. Translocation frequency determination by serial dilution 289
4. Conclusions 290
Acknowledgments 290
References 290
Chapter Thirteen: Genome Editing for Human Gene Therapy 294
1. Introduction 295
2. Genome Editing of B2M in Primary Human CD4+ T Cells 297
2.1. Required materials 298
2.2. Isolation of CD4+ T cells from peripheral blood 299
Notes 300
2.3. Delivery of CRISPR/Cas9 by nucleofection 300
2.3.1. Nucleofection 301
2.3.2. Postnucleofection 303
Notes 303
2.4. Evaluation of targeting efficiency 303
2.4.1. FACS-based analysis 304
2.4.2. PCR-based screening assay 305
Notes 306
3. Targeting of CCR5 in Human CD34+ HSPCs Using CRISPR/Cas9 307
3.1. Required materials 309
3.2. Transfection of CD34+ HSPCs 310
3.2.1. Isolation of CD34+ HSPCs from cord blood 310
3.2.2. Nucleofection of CD34+ HSPCs 311
3.2.3. Cell sorting 311
Notes 312
3.3. Colony-forming cell assay 312
Notes 313
3.4. Clonal analysis 313
Notes 314
References 314
Chapter Fourteen: Generation of Site-Specific Mutations in the Rat Genome Via CRISPR/Cas9 318
1. Theory 319
2. Equipment 321
3. Materials 322
3.1. Solutions and buffers 323
4. Protocol 324
4.1. Preparation 324
4.2. Duration 324
4.3. Caution 325
5. Step 1: In Vitro Transcription of sgRNA Target Oligonucleotides 325
5.1. Overview 325
5.2. Duration 325
5.3. Tip 326
6. Step 2: In Vitro Transcription of Cas9 mRNA 328
6.1. Overview 328
6.2. Duration 328
6.3. Tip 329
7. Step 3: Preparation of Pseudopregnant Female Rats and One-Cell Rat Embryos 330
7.1. Overview 330
7.2. Duration 330
7.3. Tip 330
7.4. Tip 331
7.5. Tip 331
8. Step 4: Microinjection of One-Cell Embryos and Transplanting the Embryos into Pseudopregnant Rats 332
8.1. Overview 332
8.2. Duration 332
8.3. Tip 333
8.4. Tip 333
8.5. Tip 333
8.6. Tip 333
8.7. Tip 333
8.8. Tip 335
9. Step 5: Identification of Founder Rats 335
9.1. Overview 335
9.2. Duration 335
9.3. Tip 336
9.4. Tip 336
10. Step 6: Production of F1 Generation Rats 338
10.1. Overview 338
10.2. Duration 338
10.3. Tips 338
References 338
Chapter Fifteen: CRISPR/Cas9-Based Genome Editing in Mice by Single Plasmid Injection 340
1. Introduction 341
2. Design and Construction of CRISPR/Cas9 Plasmids with pX330 343
2.1. Selection and off-target analysis of sgRNA in targeted gene 343
2.1.1. Design of sgRNAs against the target gene: Protocol 344
2.2. Construction of pX330 with designed sgRNA 344
2.2.1. Insertion of sgRNA into the pX330 plasmid: Protocol 344
3. Validation of pX330 In Vitro 347
3.1. Construction of pCAG-EGxxFP with the targeted genomic region 347
3.1.1. Insertion of the target genomic fragment into pCAG-EGxxFP plasmid: Protocol 348
3.2. Cotransfection of pX330-sgRNA and pCAG-EGxxFP-target into HEK293T cells 349
3.2.1. Cell culture and transfection in HEK293T cells: Protocol 349
3.3. Observation of EGFP fluorescence in the transfected cells 350
4. One-Step Generation of Mutant Mice Via Circular Plasmid Injection 351
4.1. Collecting the fertilized eggs 351
4.1.1. Superovulation treatment and collection of fertilized eggs: Protocol 351
4.2. Preparing pX330-sgRNA plasmid for microinjection 351
4.2.1. Preparation of pX330-sgRNA plasmid for microinjection: Protocol 352
4.3. Pronuclear microinjection of circular pX330-sgRNA plasmid 352
4.3.1. Manipulating mouse embryos and microinjection system: Protocol 352
5. Screening for Targeted Mutation in Mice 353
5.1. Direct sequencing of PCR products: Protocol 353
6. Concluding Remarks 353
Acknowledgment 356
References 356
Chapter Sixteen: Imaging Genomic Elements in Living Cells Using CRISPR/Cas9 358
1. Introduction 359
1.1. Choice of target sites and DNA recognition methods 359
1.2. Sensitivity and specificity of genome imaging using CRISPR/Cas9 361
2. Generation of Cell Lines Stably Expressing dCas9-GFP 362
2.1. Generation of dCas9-GFP constructs 362
2.2. dCas9-GFP/Tet-On 3G lentiviral production 363
2.3. dCas9-GFP/Tet-On 3G lentiviral infection 364
2.4. Selection of clonal cell lines stably expressing dCas9-GFP 366
3. Expression of sgRNAs Using Lentiviral Vector 367
3.1. sgRNA design and cloning 367
3.2. sgRNA lentiviral infection 368
4. Labeling of Nonrepetitive Sequences 368
4.1. Target selection and sgRNA design 368
4.2. High-throughput sgRNA cloning 369
4.3. Production of pooled sgRNA lentiviruses 370
5. Imaging of Genomic Loci Detected by CRISPR 370
5.1. Verify CRISPR signal by a modified FISH staining protocol 370
5.2. Live-cell imaging of genomic loci 371
6. Summary 373
Acknowledgments 374
References 374
Chapter Seventeen: Cas9-Based Genome Editing in Xenopus tropicalis 376
1. Introduction 377
2. Principle 378
3. Protocol 380
3.1. Background knowledge and experimental equipment 380
3.2. sgRNA design 380
3.2.1. Considerations in target site choice 382
3.3. sgRNA template construction 383
3.3.1. Template assembly by PCR: Primers 383
3.3.2. Template assembly by PCR: Assembly conditions 383
3.3.3. In vitro transcription of sgRNA 384
3.4. Procedure for microinjection 385
3.4.1. Doses of sgRNA and Cas9 385
3.4.2. Sidebar: Cas9 protein in vitro cleavage assays 388
3.4.3. Procedure for embryo microinjection 388
3.5. Assessment of mutagenesis: Genotyping 389
3.5.1. Embryo lysis and PCR 389
3.5.2. Evaluation of sequencing results and subsequent identification of specific indels 390
4. Discussion 391
4.1. Multiple targeting strategy: Avoiding off-target problems and simpler genotyping of F1 animals 391
4.2. Further applications of CRISPR-mediated mutagenesis in Xenopus 393
Acknowledgments 394
References 394
Chapter Eighteen: Cas9-Based Genome Editing in Zebrafish 398
1. Introduction 399
1.1. CRISPR/Cas adaptive immunity 399
1.2. The Type II CRISPR/Cas system 400
1.3. The development of CRISPR/Cas genome-editing technology 401
1.4. The zebrafish animal model and CRISPR/Cas 404
2. Targeted Generation of Indel Mutations 406
2.1. Cas9 modification and delivery platforms 406
Protocol for preparation of SpCas9 mRNA for microinjection 407
2.2. Single-guide RNA design considerations 409
Protocol for preparation of sgRNAs for microinjection: 413
2.3. Introduction and identification of Cas9-sgRNA-induced indels 416
3. Other Targeted Genome-Editing Strategies 417
3.1. Precise sequence modifications mediated by single-stranded oligonucleotides 417
3.2. Targeted integration of long DNA fragments 418
3.3. Chromosomal deletions and other rearrangements 421
4. Future Directions 422
Acknowledgments 424
References 424
Chapter Nineteen: Cas9-Based Genome Editing in Drosophila 436
1. Introduction 436
2. Applications and Design Considerations for CRISPR-Based Genome Editing 438
2.1. Selection of sgRNA target sites 440
2.2. Tools facilitating sgRNA design 441
3. Delivery of CRISPR Components 442
4. Generation of CRISPR Reagents 444
4.1. Cloning of sgRNAs into expression vectors 445
Materials 446
Protocol 446
4.2. Cloning of donor constructs 447
Materials 448
Protocol 449
4.3. Isolation of in vivo genome modifications 450
5. Detection of Mutations 450
5.1. Preparation of genomic DNA from fly wings 451
5.1.1. Restriction profiling 452
Materials 452
Protocol 452
5.1.2. Surveyor assay to detect indels 453
Materials 454
Protocol 454
5.1.3. Detection of mutations using HRMA 455
Materials 456
Protocol 456
5.2. Analysis of HRMA data 457
Acknowledgments 457
References 458
Chapter Twenty: Transgene-Free Genome Editing by Germline Injection of CRISPR/Cas RNA 462
1. Theory, Philosophy, and Practical Considerations 463
1.1. Overview 463
1.2. When to use or not to use transgenes for delivery of CRISPR/Cas 464
1.3. Altered mutation profile from transgene-free treatment with CRISPR/Cas 465
1.4. A note on specificity of CRISPR/Cas cleavage 467
2. Equipment 467
3. Materials 468
4. Identifying a Target Sequence 468
5. Generating Your sgRNA Construct 470
5.1. Oligonucleotide design 470
5.2. Insert generation 470
5.3. Preparation of linearized vector for the sgRNA construct 471
5.4. Construction and identification of sgRNA synthesis plasmid 471
6. In Vitro Synthesis of sgRNA 472
6.1. Linearization of sgRNA template plasmid 472
6.2. In vitro transcription to generate sgRNA 473
6.3. Purification of in vitro-transcribed sgRNA 473
7. In Vitro Synthesis of hCas9 mRNA 473
7.1. Linearization of SP6-hCas9-Ce-mRNA plasmid 473
7.2. In vitro transcription of hCas9 mRNA 474
7.3. Polyadenylation of in vitro-transcribed hCas9 mRNA 474
7.4. Purification of in vitro-transcribed, polyadenylated hCas9 mRNA 474
8. Injection of sgRNA and mRNA 474
9. Recovery of Mutants Generated Using CRISPR/Cas 475
9.1. Recovery and plating of injected animals 475
9.2. Identification of animals carrying mutations induced by CRISPR/Cas 476
References 476
Chapter Twenty-One: Cas9-Based Genome Editing in Arabidopsis and Tobacco 480
1. Introduction 481
2. Cas9 and sgRNA expression 482
3. Dual sgRNA-Guided Genome Editing 484
3.1. Designing and constructing dual sgRNAs 484
3.2. Transfecting and expressing Cas9/sgRNAs in protoplasts 485
3.3. Evaluating the frequency of targeted genome modifications 486
4. Perspectives 488
5. Notes 489
Acknowledgments 491
References 491
Chapter Twenty-Two: Multiplex Engineering of Industrial Yeast Genomes Using CRISPRm 494
1. Introduction 495
2. Plasmid Design 497
3. Cas9 Expression 499
4. Guide RNA Expression 499
5. Screening Method 502
5.1. Cloning the target sequence into pCAS 503
5.2. Double-stranded linear DNA repair oligos 503
5.3. CRISPRm screening consists of the cotransformation of pCAS and the double-stranded linear DNA homologous repair template 504
5.4. Industrial yeast 506
5.5. Markerless gene assembly in the yeast chromosome 506
6. Concluding Remarks 508
Acknowledgments 509
References 509
Chapter Twenty-Three: Protein Engineering of Cas9 for Enhanced Function 512
1. Introduction 513
1.1. The structure of Cas9 515
1.2. Current uses 518
1.3. Initial engineering questions 518
2. Methods 519
2.1. A note on applications 519
2.2. Electrocompetent E. coli preparation for library construction 520
2.3. Discovery of functional, engineered, variants of Cas9 proteins 521
2.4. Screening Cas9 521
2.5. Selecting Cas9 521
2.6. Screening for functional Cas9 variants 523
2.7. Determining screening enrichment of PDZ-dCas9 domain insertions 525
2.8. Identifying and testing PDZ-Cas9 clones from a screened library 527
2.9. Expanding horizons 528
3. Conclusion 529
References 529
Author Index 534
Subject Index 560
Color Plate 571
In Vitro Enzymology of Cas9
Carolin Anders; Martin Jinek1 Department of Biochemistry, University of Zurich, Zurich, Switzerland
1 Corresponding author: email address: jinek@bioc.uzh.ch
Abstract
Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9–guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.
Keywords
CRISPR
crRNA
tracrRNA
Endonuclease
Protein–RNA complex
Genome editing
Gene targeting
RNA guided
Biochemical assay
1 Introduction
The clusters of regularly interspaced short palindromic repeat (CRISPR)-associated protein Cas9 is an RNA-guided endonuclease that generates double-strand DNA breaks (DSBs) (reviewed in Hsu, Lander, & Zhang, 2014; Mali, Esvelt, & Church, 2013). Found in type II CRISPR systems, Cas9 functions in conjunction with CRISPR RNAs (crRNAs) and a transactivating crRNA (tracrRNA) to mediate sequence-specific immunity against bacteriophages and other mobile genetic elements (Barrangou et al., 2007; Deltcheva et al., 2011; Garneau et al., 2010). Cas9 associates with a partially base-paired crRNA–tracrRNA guide structure and the resulting ribonucleoprotein complex recognizes and cleaves DNA molecules containing sequences complementary to a 20-nucleotide guide segment in the crRNA (Gasiunas, Barrangou, Horvath, & Siksnys, 2012; Jinek et al., 2012; Karvelis et al., 2013).
Due to its programmability, Cas9 has been developed into a versatile molecular tool for genome editing in numerous organisms and cell types (reviewed extensively in Hsu et al., 2014; Mali, Esvelt, et al., 2013; Sander & Joung, 2014), including human cells (Cong et al., 2013; Jinek et al., 2013; Mali, Yang, et al., 2013), mice (Wang et al., 2013; Yang et al., 2013), zebrafish (Hwang et al., 2013), Drosophila melanogaster (Bassett & Liu, 2014; Gratz et al., 2013), Caenorhabditis elegans (Cho, Lee, Carroll, Kim, & Lee, 2013; Friedland et al., 2013; Katic & Grosshans, 2013; Lo et al., 2013), and plants (Li et al., 2013; Nekrasov, Staskawicz, Weigel, Jones, & Kamoun, 2013; Shan et al., 2013; Xie & Yang, 2013). The sequence specificity of Cas9 permits the targeting of unique loci in a typical eukaryotic genome and can be readily altered in vitro and in vivo by supplying artificially designed guide RNAs either in the naturally occurring dual-RNA form or as single-molecule guide RNAs (sgRNAs) (Cong et al., 2013; Jinek et al., 2012, 2013; Mali, Yang, et al., 2013). Cas9 thus provides a superior alternative to existing protein-based approaches such as zinc finger nucleases and transcription activator-like effector nucleases. In eukaryotic cells, Cas9-generated DSBs are repaired by nonhomologous end joining or homologous recombination, which can be exploited to engineer insertions, deletions, and substitutions in the vicinity of the DSB. Furthermore, a catalytically inactive variant of Cas9 (the D10A/H840A mutant of Streptococcus pyogenes Cas9, referred to as dCas9) has been employed as an RNA-programmable DNA-binding protein for transcriptional regulation (Gilbert et al., 2013; Mali, Aach, et al., 2013; Qi et al., 2013). Variants of the basic targeting approach, including paired nickases (Mali, Aach, et al., 2013; Ran et al., 2013), dCas9-FokI fusion nucleases (Guilinger, Thompson, & Liu, 2014; Tsai et al., 2014), and 5′-truncated sgRNAs (Fu, Sander, Reyon, Cascio, & Joung, 2014) have emerged recently to address the issue of off-targeting and to further improve Cas9 specificity.
Extensive biochemical and structural studies have illuminated many aspects of the molecular mechanism of Cas9. The two nuclease domains found in Cas9, HNH and RuvC domains, catalyze the cleavage of the complementary and noncomplementary DNA strands, respectively (Chen, Choi, & Bailey, 2014; Gasiunas et al., 2012; Jinek et al., 2012). Target DNA recognition is strictly dependent on the presence of a short protospacer adjacent motif (PAM) immediately downstream of the DNA region base-paired to the guide RNA (Gasiunas et al., 2012; Jinek et al., 2012). An 8–12 nt PAM-proximal “seed” region in the guide RNA–target DNA heteroduplex is critical for target binding by Cas9 (Jinek et al., 2012; Nishimasu et al., 2014). While seed region interactions are sufficient for target binding, DNA cleavage requires more extensive guide–target interactions (Wu et al., 2014). Nevertheless, Cas9 tolerates mismatches within the guide–target heteroduplex, which is the principal cause of off-target activity (Fu et al., 2013; Hsu et al., 2013; Mali, Aach, et al., 2013; Pattanayak et al., 2013). Recent crystal structures and electron microscopic reconstructions of Cas9 in its free and nucleic-acid-bound states have revealed that Cas9 undergoes a striking RNA-driven conformational rearrangement that results in the formation of the DNA-binding site (Anders, Niewoehner, Duerst, & Jinek, 2014; Jinek et al., 2014; Nishimasu et al., 2014). Additionally, single-molecule and ensemble biophysical studies of target recognition by the Cas9–guide RNA complex have indicated that target DNA binding is dependent on an initial recognition of the PAM, followed by local unwinding of the adjacent DNA duplex and directional formation of the guide RNA–target DNA heteroduplex (Sternberg, Redding, Jinek, Greene, & Doudna, 2014).
In this Chapter, we provide detailed protocols for the heterologous expression and purification of S. pyogenes Cas9, preparation of guide RNAs by in vitro transcription, and for the use of these reagents in endonuclease cleavage assays in vitro. The assays described here can be used to validate guide RNAs and target sites for in vivo gene targeting applications or to test the in vitro efficacy of new guide RNA structures and designs. Moreover, the described procedures can be implemented to utilize Cas9 as a programmable restriction enzyme for DNA manipulations in vitro. Although S. pyogenes Cas9 has been the mainstay of genome editing applications so far, the protocols are readily adaptable for Cas9 proteins and guide RNAs from other bacterial species and may aid in the rational design of novel Cas9 variants with altered specificity or PAM requirements.
2 Expression and Purification of Cas9
Cas9 from S. pyogenes (hereafter referred to as SpyCas9) is expressed from a pET-based T7 promoter-containing plasmid (pMJ806, available from Addgene, www.addgene.org) in the E. coli strain Rosetta 2 DE3. The expressed fusion protein construct contains an N-terminal His6-tag, followed by maltose-binding protein (MBP) polypeptide sequence, a tobacco etch virus (TEV) protease cleavage site, and the SpyCas9 sequence spanning residues 1–1368. We found that expression in the Rosetta 2 strain was necessary to overcome the unfavorable codon bias in the S. pyogenes genomic DNA sequence, while inclusion of the MBP tag further boosted expression levels. The purification protocol includes three chromatography steps: immobilized metal ion affinity chromatography (IMAC), followed by cation exchange chromatography (IEX), and a final purification by size exclusion chromatography (SEC). The protocol is generally based on previously published procedures, with minor modifications (Jinek et al., 2012, 2014; Sternberg et al., 2014). The procedure can be used for the expression and purification of mutant SpyCas9 proteins and can be adapted for the expression of Cas9 orthologs from other bacterial species.
Day 1: Cell transformation
1. Transform chemically competent Rosetta 2 DE3 cells (Novagen, Merck Millipore) according to the protocol supplied with the cells. Briefly, add ~ 200 ng of plasmid DNA (pMJ806) to 50 μl of freshly thawed competent cells and incubate on ice for 15 min. Heat-shock cells by incubation at 42 °C for 45 s, then place cells on ice for further 3 min. Add 500 μl of LB (Luria Broth) medium to the cells and incubate the culture at 37 °C for 1 h in a shaking incubator. Plate 100 μl of culture out on LB agar containing 50 μg ml− 1 kanamycin and...
| Erscheint lt. Verlag | 4.11.2014 |
|---|---|
| Sprache | englisch |
| Themenwelt | Medizin / Pharmazie |
| Naturwissenschaften ► Biologie ► Biochemie | |
| Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie | |
| Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik | |
| ISBN-10 | 0-12-801334-6 / 0128013346 |
| ISBN-13 | 978-0-12-801334-2 / 9780128013342 |
| Haben Sie eine Frage zum Produkt? |
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