CRISPR-Cas methods [Volume 1

This volume details the fundamentals of the CRISPR-Cas system, and its protocols illustrate advances in CRISPR-Cas techniques for efficient genome editing. Introductory chapters provide a wide horizon of CRISPR/Cas-based methods and applications. Additional chapters guide readers through HDR-mediated editing, sgRNA design, the step-by-step procedure of multiplex adenine base editing experiments in rice, generating mutants for rice, wheat, Brachypodium, Barley, Flax, and Phytophthora, visual screening of mutants, gene deletion (knock-out), tagging (knock-in) in mammalian cells, the cloning-free (DNA-free) technique, cell-penetrating peptides, generating a genome-edited banana, and nuclear genome editing of Chlamydomonas employing CRISPR-Cpf1 combined with a single-stranded DNA (ssODN) repair template. Authoritative and cutting-edge, CRISPR-Cas Methods aims to assist researchers who are new to the field and are aiming to learn how best to adopt this technology for a particular organism. Foreword......Page 6 Preface......Page 7 Contents......Page 10 Contributors......Page 12 1 Basics of CRISPR-Cas Immune System......Page 14 2 Development of Genome Editing Tools from the Prokaryotic Immune System......Page 15 3 Orthologous and Engineered Cas Proteins for Diverse Genome Editing Platform......Page 16 4 CRISPR-Cas Tool Box Has Too Much to Consider......Page 19 4.2 Gene Regulation......Page 23 4.4 Epigenome Editing......Page 25 4.6 Chromatin Topology......Page 26 4.8 Base Editing......Page 27 4.9 RNA Editing......Page 29 4.11 CRISPR-Mediated Homology-Directed Repair (HDR)......Page 30 4.13 Molecular Diagnostic......Page 31 5 Concluding Remarks......Page 32 References......Page 33 1 Introduction......Page 37 2.1.1 pDe-SaCas9-EC......Page 39 2.3 Reagents......Page 40 3.2 Generating the Required T-DNAs......Page 41 3.3 Obtaining Heritable GT Plants (See also Fig. 2)......Page 42 4 Notes......Page 43 References......Page 45 Abbreviations......Page 47 1 Introduction......Page 48 2.1.1 Sequence Information Acquisition......Page 49 2.2.2 Protoplast Assay......Page 50 2.2.3 Indica Rice Transformation......Page 51 3.1.3 Cloning......Page 52 3.2.1 Protoplast Isolation (See Note 10)......Page 53 3.3.2 Co-cultivation: Immature Embryo Transformation......Page 54 3.4.2 Screening and Validation of Variation in Genomic Region of Interest......Page 55 4 Notes......Page 56 References......Page 61 1 Introduction......Page 63 2.1 Plasmid Vectors......Page 64 2.2.2 Rice Transformation......Page 66 3.2 Designing of Primers......Page 67 3.3 Assembly of Multiple sgRNAs through Golden Gate Assembly......Page 68 3.4 Cloning of sgRNAs into the Base Editor (BE) Vector......Page 70 3.7 Selection and Regeneration......Page 71 3.8 Detection of Base Editing......Page 72 References......Page 73 1 Introduction......Page 75 2.1.2 Materials for Transformation and Regeneration of Brachypodium distachyon......Page 77 3.1 Cloning of Single gRNA into pRGEB32......Page 79 3.2 Cloning of Two Guide RNAs Spaced Between Two tRNAs to Boost Cas9 Efficiency (tRNA-gRNA3-tRNA-gRNA4) into pRGEB32......Page 81 4 Sequencing of CRISPR Constructs......Page 87 6 Transformation of Agrobacterium tumefaciens AGL-1 by Heat Shock......Page 89 7 Generation of Brachypodium distachyon Embryogenic Callus......Page 90 8 Agrobacterium Infection of Brachypodium Callus......Page 93 9 Regeneration of Transformed Brachypodium Plants......Page 94 11 Notes......Page 96 References......Page 98 1 Introduction......Page 99 2.1 sgRNA Design and Cloning......Page 100 2.4 Media and Supplies for P. palmivora Growth and Transformation......Page 101 3.1.1 Selection of 20-Nucleotide (nt) sgRNA Target Sequences......Page 103 3.1.2 Cloning of sgRNA Target Sequences......Page 104 3.2.2 Preparation of Phytophthora palmivora Zoospores......Page 105 3.2.4 Selection of G418-Resistant Transformants......Page 106 3.4.1 Isolate Genomic DNA from Transformants Using DNeasy PowerLyzer Microbial Kit (Qiagen)......Page 107 4 Notes......Page 108 References......Page 109 1 Introduction......Page 111 2.1 Plant and Bacteria Material......Page 112 2.5 B. napus Tissue Culture Media......Page 113 3.1 Selection of sgRNA Targeting Sequences and Design of Target Adaptors......Page 114 3.2 Isolation of the Binary Plasmids and sgRNA Intermediate Plasmids......Page 115 3.3 Assembly of sgRNA Expression Cassettes into a CRISPR-Cas9 Binary Construct......Page 117 3.4 Transformation of E. coli and A. tumefaciens Strains......Page 122 3.5 Plant Transformation......Page 123 3.6 Sequencing Analysis of Targeted Mutations in Transgenic Plants......Page 124 4 Conclusions......Page 125 References......Page 126 1 Introduction......Page 128 2.2 Prediction of off-Target Sites......Page 131 2.4 Cloning......Page 132 2.5.2 PCR and Electrophoresis......Page 133 3.1 Design of the Cas9 Gene......Page 134 3.2 Selection of Target Sequences......Page 135 3.4 Assembly of the sgRNA Construct......Page 138 3.5 Assembly of the PTG Construct for Multiplex Genome Editing......Page 142 3.6.1 DNA Extraction from Barley Plants (See Note 2)......Page 143 3.6.3 Restriction Analysis of PCR Products......Page 144 3.6.4 T7 Endonuclease I Cleavage Method......Page 145 3.6.5 qPCR with Dual Labeled Molecular Probes......Page 147 3.7 Identification of T1 Transgene-Free Mutant Lines......Page 148 4 Notes......Page 150 References......Page 152 1 Introduction......Page 154 2.3 Delivering Foreign DNA Inside Cells......Page 157 3.1 Choosing a Gene-Targeting Strategy......Page 158 3.2.2 gRNA Synthesis and Cloning into the Delivery Vector......Page 159 Electroporation for Noncancerous Cells (RPE1)......Page 162 3.2.5 Determining the Cutting Efficiency of gRNA......Page 163 Surveyor Assay/CEL-1 Assay......Page 165 Base Sequence Mix-Read Assay......Page 167 3.3.1 Ordering Oligos and Necessary Plasmids......Page 168 3.3.2 Generating PCR Cassette......Page 169 3.4 Validating the Genome Modification......Page 171 References......Page 172 1 Introduction......Page 174 2.2 Complete Medium for THLE-2 Cell Line......Page 175 3.3 Preparation of gRNA for Cell Transfection......Page 176 3.5 Preparation of THLE-2 Cells......Page 177 3.8 Direct DNA Amplification......Page 178 3.9 T7 Endonuclease I Digestion......Page 179 4 Notes......Page 180 References......Page 181 1 Introduction......Page 183 2.1 Identification of gRNA......Page 184 2.2 Validation of gRNA......Page 185 2.2.1 Digestion Using Cas9 Nuclease......Page 187 2.4.1 Primers Designing......Page 188 2.4.2 Gibson Assembly......Page 190 2.4.3 Chemically Competent Bacterial Cell Transformation......Page 192 2.5 Plant Material and Protoplast Isolation......Page 193 2.5.2 Protoplast Isolation......Page 194 2.5.3 Protoplast Transfection and Culture......Page 195 2.7 Detection of Induced Mutation Using T7E1 Assay and DNA Sequencing......Page 196 2.7.2 Mutation Confirmation by Sequencing......Page 197 3 Notes......Page 198 References......Page 199 1 Introduction......Page 201 2.2 Components Required for Cloning and Gene Expression Analysis......Page 203 2.5 Cell-Penetrating Peptide and Protein Labeling......Page 204 3.1 Overexpression and Purification of Cas9 and dCas9-VP64 Proteins from Bacterial Culture Using FPLC System......Page 205 3.2 Isolation of Wheat Microspores......Page 206 3.3 In Vitro Transcription of sgRNA and Testing Activity of Cas9/sgRNA RNP......Page 207 3.4 Labeling of Cas9 Protein with the Fluorophore and Transfection of Microspores with RNP-CPP Complex......Page 208 4 Notes......Page 211 References......Page 212 1 Introduction......Page 213 2 Designing of Guide RNA......Page 214 3 Cloning and Reassembling Constructs......Page 218 4.1 Mesophyll Protoplast System......Page 224 5 Wheat Transformation......Page 226 5.1 Microspore Transformation Using......Page 227 6 Transformation of Wheat Embryos Using Biolistic Gen Gun......Page 228 7.1 Microspore Regeneration After Electroporation......Page 229 7.2 Embryo Regeneration After Bombardment......Page 230 References......Page 231 1 Introduction......Page 233 2.2 Tools for Designing gRNA......Page 234 2.3 Plasmid Constructs......Page 235 2.5 Banana Protoplast......Page 236 3.3 Preparation of CRISPR-Cas9 Ribonucleoproteins (RNPs)......Page 237 3.4 Delivery of Plasmid Construct into Banana Cells and Generation of Complete Plants......Page 239 3.5 Delivery of CRISPR-Cas9 Ribonucleoprotein (RNP) Complexes into Banana Protoplast and Regeneration of Complete Plants......Page 240 3.6.4 T7 Analysis......Page 241 3.6.6 Evaluation of Off-Target Mutations......Page 242 3.7.2 Phenotyping for BXW......Page 243 References......Page 244 1 Introduction......Page 247 2.1 TAP Medium......Page 248 2.3 C. reinhardtii Electroporation......Page 250 2.4 Cell Plating......Page 251 3.1 Chlamydomonas reinhardtii Culture......Page 252 3.2 Electroporation......Page 253 3.5 Colony PCR......Page 254 4 Notes......Page 255 References......Page 261 Glossary......Page 263 References......Page 282 Index......Page 283