| Contributors |
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| Preface |
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xvii | |
| 1 Predicting and visualizing features of CRISPR-Cas systems |
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1 | (26) |
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2 | (2) |
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2 Identify and characterize CRISPR-Cas system enzymes |
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4 | (6) |
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3 Visualize and characterize the repeat-spacer array |
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10 | (2) |
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12 | (4) |
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5 Predicting the PAM sequence |
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16 | (4) |
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20 | (1) |
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20 | (7) |
| 2 Reconstitution and biochemical characterization of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems |
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27 | (16) |
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28 | (4) |
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2 Electrophoretic mobility shift assay |
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32 | (7) |
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39 | (1) |
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39 | (1) |
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40 | (1) |
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41 | (2) |
| 3 Sortase-mediated fluorescent labeling of CRISPR complexes |
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43 | (18) |
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44 | (3) |
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47 | (8) |
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55 | (2) |
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57 | (1) |
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57 | (1) |
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57 | (1) |
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57 | (4) |
| 4 Fluorescence-based methods for measuring target interference by CRISPR-Cas systems |
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61 | (26) |
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62 | (2) |
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2 Fluorescence-based strategies for measuring CRISPR interference |
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64 | (5) |
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3 Measurement of CRISPR interference in colonies and liquid culture |
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69 | (8) |
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4 Measuring CRISPR interference from a plasmid-borne Cas effector |
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77 | (4) |
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81 | (1) |
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81 | (1) |
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81 | (6) |
| 5 Probing Cascade complex composition and stability using native mass spectrometry techniques |
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87 | (30) |
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Monika Tokmina-Lukaszewska |
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88 | (2) |
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2 Native mass spectrometry |
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90 | (16) |
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106 | (4) |
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110 | (1) |
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111 | (1) |
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111 | (6) |
| 6 High-throughput determination of in vivo DNA sequence preferences for Cas protein binding using Library-ChIP |
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117 | (16) |
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118 | (1) |
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2 Library-ChIP of Cas proteins |
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118 | (13) |
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131 | (1) |
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132 | (1) |
| 7 Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance |
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133 | (40) |
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134 | (7) |
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2 Generation of Cascade complexes with a fluorescent tag |
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141 | (8) |
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3 Heterologous expression of Cascade |
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149 | (4) |
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4 sptPALM imaging of Cascade interference |
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153 | (12) |
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165 | (1) |
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165 | (1) |
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165 | (8) |
| 8 In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system |
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173 | (18) |
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174 | (2) |
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2 Strategy for purification of soluble subunits |
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176 | (2) |
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3 Expression and purification of recombinant subunits and subcomplexes |
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178 | (6) |
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4 In vitro transcription of crRNA |
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184 | (2) |
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5 In vitro assembly of ToCsm complexes |
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186 | (2) |
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188 | (1) |
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188 | (1) |
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188 | (3) |
| 9 Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems |
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191 | (28) |
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192 | (2) |
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2 Expression and purification of CARF proteins in Escherichia coli |
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194 | (6) |
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3 Generating oligoadenylates for CARF protein activation |
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200 | (8) |
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4 Breakdown of cyclic oligoadenylates |
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208 | (3) |
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5 Characterization of reaction products by thin-layer chromatography and mass spectrometry |
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211 | (5) |
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216 | (1) |
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216 | (1) |
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216 | (3) |
| 10 A pipeline for characterization of novel Cas9 orthologs |
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219 | (22) |
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220 | (3) |
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223 | (12) |
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235 | (1) |
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235 | (1) |
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236 | (5) |
| 11 Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells |
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241 | (24) |
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242 | (2) |
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2 Expression and purification of the Cas12a-NLS-NLS-eGFP-NLS fusion protein |
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244 | (2) |
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3 Design of crRNA guides for introducing gene deletions into the mouse genome |
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246 | (1) |
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4 Verification of DNA cleavage activity in vitro |
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246 | (6) |
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5 Electroporation of RNP complexes into mouse ES cells |
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252 | (3) |
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6 Enrichment of eGFP-positive ES cells by fluorescence-activated cell sorting |
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255 | (1) |
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7 PCR-and 17 endonuclease-based assays for detecting deletions and mutations in ES cell pools |
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255 | (4) |
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8 Examples of Cas12a-mediated Ubn1, Ubn2, and Rbm12 gene deletions in mouse ES cells |
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259 | (1) |
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260 | (1) |
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261 | (4) |
| 12 Directed evolution studies of a thermophilic Type II-C Cas9 |
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265 | (24) |
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266 | (5) |
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2 Characterization of AceCas9 |
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271 | (5) |
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3 Directed evolution of AceCas9 |
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276 | (5) |
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4 Functional regions of AceCas9 identified by directed evolution |
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281 | (3) |
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284 | (1) |
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284 | (1) |
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284 | (5) |
| 13 Kinetic characterization of Cas9 enzymes |
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289 | (24) |
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290 | (2) |
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2 Preparation of Cas9 and guide RNA complexes |
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292 | (2) |
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3 General cleavage assay protocol |
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294 | (1) |
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4 Active-site titration assay |
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294 | (4) |
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298 | (7) |
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305 | (1) |
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7 Analyzing data using KinTek Explorer |
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306 | (3) |
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309 | (1) |
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309 | (1) |
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309 | (1) |
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309 | (4) |
| 14 Single-molecule FRET studies of Cas9 endonuclease |
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313 | (24) |
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314 | (2) |
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2 TIRF-based single-molecule FRET |
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316 | (3) |
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3 Preparation for single-molecule experiments |
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319 | (2) |
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4 DNA immobilization-based assays |
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321 | (5) |
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5 SpCas9 immobilization-based assays |
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326 | (5) |
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331 | (2) |
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333 | (1) |
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333 | (1) |
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333 | (4) |
| 15 CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA |
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337 | (28) |
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338 | (2) |
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2 Design and validation of Cas beacon method |
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340 | (4) |
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3 Studies of assembly of CRISPR-Cas effector complexes |
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344 | (10) |
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4 Determination of CRISPR-Cas effectors affinities for DNA probes |
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354 | (6) |
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360 | (1) |
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360 | (1) |
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360 | (5) |
| 16 Adapting dCas9-APEX2 for subnuclear proteomic profiling |
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365 | (20) |
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366 | (1) |
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2 Generation of cell lines stably expressing dCas9-APEX2 and sgRNAs |
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367 | (4) |
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3 Assessing dCas9-APEX2-specific DNA targeting and biotin-labeling signals at repetitive loci |
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371 | (6) |
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4 SILAC labeling, C-BERST biotinylation, and enrichment of biotinylated proteins |
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377 | (2) |
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5 Mass spectrometry analysis |
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379 | (2) |
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6 Further configuration of C-BERST |
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381 | (1) |
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381 | (1) |
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382 | (3) |
| 17 CRISPR-Cas10 assisted editing of virulent staphylococcal phages |
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385 | (26) |
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386 | (3) |
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2 Phage targeting in S. epidermidis |
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389 | (9) |
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3 Phage editing in S. epidermidis |
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398 | (5) |
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4 Editing phages that infect CRISPR-less Staphylococcus hosts |
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403 | (2) |
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5 Transforming Staphylococcus bacteria |
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405 | (2) |
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407 | (1) |
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407 | (1) |
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408 | (3) |
| 18 Reconstitution of CRISPR adaptation in vitro and its detection by PCR |
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411 | |
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412 | (1) |
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2 Cas1:Cas2-3 complex purification |
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413 | (8) |
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421 | (4) |
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4 Spacer integration assay and detection by PCR |
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425 | (6) |
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431 | (1) |
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431 | (1) |
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431 | |
