CRISPR-Cas enzymes / edited by Scott Bailey.

Format:

Book

Contributor:

Bailey, Scott, editor.

Series:

Methods in enzymology ; Volume 616.

Methods in enzymology ; Volume 616

Language:

English

Subjects (All):

CRISPR (Genetics).

Physical Description:

1 online resource (xviii, 433 pages) : illustrations.

Place of Publication:

Cambridge, Massachusetts : Academic Press, [2019]

Summary:

CRISPR-Cas Enzymes, Volume 616, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Topics covered in this release include CRISPR bioinformatics, A method for one-step assembly of Class 2 CRISPR arrays, Biochemical reconstitution and structural analysis of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems, Mechanistic dissection of the CRISPR interference pathway in Type I-E CRISPR-Cas system, Site-specific fluorescent labeling of individual proteins within CRISPR complexes, Fluorescence-based methods for measuring target interference by CRISPR-Cas systems, Native State Structural Characterization of CRISRP Associated Complexes using Mass Spectrometry, and more.- Provides the authority and expertise of leading contributors from an international board of authors- Presents the latest release in the Methods in Enzymology series- Updated release includes the latest information on the CRISPR-Cas Enzymes

Contents:

Front Cover

CRISPR-Cas Enzymes

Copyright

Contents

Contributors

Preface

Chapter One: Predicting and visualizing features of CRISPR-Cas systems

1. Introduction

2. Identify and characterize CRISPR-Cas system enzymes

2.1. Requirements

2.2. Metagenomic preparation

2.3. Annotation

2.4. CRISPR-Cas system identification and typing

3. Visualize and characterize the repeat-spacer array

3.1. Requirements

3.2. Extract and visualize the repeat spacer array

3.3. Determine CRISPR locus orientation

4. crRNA guides

4.1. Requirements

4.2. tracrRNA identification

4.3. crRNA boundaries and tracrRNA identification through RNASeq

5. Predicting the PAM sequence

5.1. Requirements

5.2. Procedure

Acknowledgments

References

Chapter Two: Reconstitution and biochemical characterization of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems

1.1. In vivo assay for Cascade- and Cas3-mediated target plasmid loss

1.2. Cloning, expression, and purification of the T. fusca Cascade complex

2. Electrophoretic mobility shift assay

2.1. Chemical probing

2.2. Assemble TfuCascade/seed-bubble, TfuCascade/R-loop, and TfuCascade/R-loop/Cas3 complexes for structural analysis

3. Conclusion

Further reading

Chapter Three: Sortase-mediated fluorescent labeling of CRISPR complexes

2. Methods

2.1. Overview

2.2. Buffers

2.2.1. Sortase purification buffers

2.2.2. Cas1-Cas2 purification buffers

2.2.3. Cascade purification buffers

2.2.4. Cas3 purification buffers

2.3. Sortase purification

2.4. Purification of Tfu Cascade complexes and subunits for N-terminal sortase labeling

2.4.1. Cas1-Cas2 purification and sortase labeling of Cas2

2.4.2. Cascade purification and sortase labeling of Cse1.

2.5. Purification of Tfu Cascade complexes and subunits for C-terminal sortase labeling

2.5.1. Cas3 purification and sortase labeling

2.6. Optimization of sortase-mediated fluorescent labeling

3. Applications

3.1. Single-molecule imaging of fluorescent Cas1-Cas2, Cascade, and Cas3 on DNA curtains

4. Notes

Funding

Chapter Four: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

2. Fluorescence-based strategies for measuring CRISPR interference

2.1. Design and development of GFP-reporter plasmid pACYC-GFP

2.2. Validation of GFP-based plasmid-loss assay

3. Measurement of CRISPR interference in colonies and liquid culture

3.1. Addition of CRISPR target to pACYC-GFP

3.1.1. Equipment

3.1.2. Buffers and reagents

3.1.3. Procedure

3.2. Detection of plasmid levels in bacterial colonies

3.2.1. Equipment

3.2.2. Buffers and reagents

3.2.3. Procedure

3.3. Measurement of CRISPR interference efficiency in liquid cultures

3.3.1. Equipment

3.3.2. Buffers and reagents

3.3.3. Procedure

4. Measuring CRISPR interference from a plasmid-borne Cas effector

4.1. Cas9 fluorescence-based plasmid loss assay

4.1.1. Equipment

4.1.2. Buffer and reagents

4.1.3. Procedure

5. Summary and conclusion

Chapter Five: Probing Cascade complex composition and stability using native mass spectrometry techniques

2. Native mass spectrometry

2.1. Preparation of capillaries for static, nanoflow electrospray ionization applications

2.1.1. Equipment

2.1.2. Procedure

2.1.3. Notes

2.2. Sample preparation for noncovalent mass spectrometry experiments

2.2.1. Equipment

2.2.2. Buffers and reagents

2.2.3. Procedure

2.2.4. Notes.

2.3. MS protocol and data analysis

2.3.1. Equipment

2.3.2. Buffers and reagents

2.3.3. Procedure

2.3.4. Notes

3. Intact protein HDX

3.1. Equipment

3.2. Buffers and reagents

3.3. Procedure

3.4. Notes

4. Summary/Conclusions

Chapter Six: High-throughput determination of in vivo DNA sequence preferences for Cas protein binding using Library-ChIP

2. Library-ChIP of Cas proteins

2.1. Construction of a plasmid library of protospacervariants

2.1.2. Buffers and reagents

2.1.3. Procedure

2.1.4. Notes

2.2. (Optional) associate protospacer variants with barcodesequences

2.2.4. Notes

2.3. Preparation of sonicated cell lysates for cells containing the protospacer library

2.4. Immunoprecipitation of Cascade from sonicated cell lysates

2.4.1. Equipment

2.4.2. Buffers and reagents

2.4.3. Procedure

2.4.4. Notes

2.5. Library amplification

2.5.1. Equipment

2.5.2. Buffers and reagents

2.5.3. Procedure

2.5.4. Notes

2.6. Sequence libraries, calculate relative enrichment values

2.6.1. Equipment

2.6.2. Procedure

2.6.3. Notes

3. Conclusions

Chapter Seven: Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance

1.1. Dynamics of Cas protein-mediated DNA targeting

2. Generation of Cascade complexes with a fluorescent tag

2.1. Target selection and oligonucleotide design for the cloning of CRISPR arrays

2.1.1. Control spacer with no targets on the genome

2.1.2. Targeting spacer with full complementarity to the host genome.

2.1.3. Targeting spacer with reduced complementarity to regions of the genome

2.2. Oligonucleotide design for the cloning of cas genes

2.3. Plasmid generation

3. Heterologous expression of Cascade

3.1. Transformation into BL21-AI

3.1.1. Generation of strains for the evaluation of Cascade activity

3.1.2. Generation of strains for sptPALM imaging

3.2. Evaluation of the fluorescent signal of Cascade-Dendra2-T69A

3.3. Evaluation of Cascade activity by efficiency of plaquing assays

4. sptPALM imaging of Cascade interference

4.1. Sample preparation for sptPALM imaging

4.2. Microscope settings and sptPALM imaging routines

4.2.1. Measuring transient Cascade-target interactions at a high spatiotemporal resolution

4.2.2. Measuring bound times of DNA-Cascade interference

4.3. Localization and tracking routines

4.3.1. Cell segmentation

4.3.2. Localization

4.3.3. Tracking

4.3.4. Data quality assessment

4.4. Data analysis and visualization

4.4.1. Obtaining molecule mobilities

4.4.2. Extracting molecular bound times

5. Conclusions

Chapter Eight: In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system

2. Strategy for purification of soluble subunits

2.1. Cloning for soluble expression of Csm subunits

2.2. Equipment and reagents

3. Expression and purification of recombinant subunits and subcomplexes

3.1. Csm3 subunit

3.2. Cas10/Csm4 subcomplex

3.3. Csm2/Csm5 subcomplex

3.4. Equipment and reagent

4. In vitro transcription of crRNA

4.1. Equipment and reagents

5. In vitro assembly of ToCsm complexes

5.1. Equipment and reagents

6. Conclusion

Chapter Nine: Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.

1. Introduction

1.1. The type III CRISPR effector complex: Targeting RNA has consequences

2. Expression and purification of CARF proteins in Escherichia coli

2.1. Subcloning genes for expression in E. coli

2.1.1. Key reagents

2.2. Protein expression

2.2.1. Mini-scale expression trials

2.2.1.1. Procedure

2.2.2. Automating protein expression screening with the BioSprint 15 (QIAGEN)

2.2.2.1. Key reagents

2.2.2.2. Procedure

2.2.3. SDS-PAGE to determine protein expression and solubility

2.2.3.1. Procedure

2.2.4. Large-scale expression

2.2.4.1. Key Reagents

2.2.4.2. Procedure

3. Generating oligoadenylates for CARF protein activation

3.1. Enzymatic cOA synthesis by a type III CRISPR complex

3.1.1. Procedure

3.2. Addressing challenges of cOA synthesis

3.3. Generating cOA analogues using the E. coli MazF nuclease

3.3.1. Purification of recombinant MazEF fusion protein

3.3.1.1. Key reagents

3.3.1.2. Procedure

3.3.2. Production of linear cOA analogues using MazF nuclease

3.3.2.1. Key reagents

3.3.2.2. Procedure

3.3.3. Activation of CARF ribonucleases by cyclic and linear oligoadenylates

3.3.3.1. Key reagents

3.3.3.2. Procedure

4. Breakdown of cyclic oligoadenylates

4.1. Ring nucleases

4.2. Kinetic analysis of cOA degradation

4.2.1. Key reagents

4.2.2. Procedure

5. Characterization of reaction products by thin-layer chromatography and mass spectrometry

5.1. Thin-layer chromatography to identify circular and linear polynucleotide species

5.1.1. Key reagents

5.1.2. Procedure

5.2. Characterization of cOA synthesis and degradation products by liquid chromatography-high-resolution mass spectrometry

5.2.1. Key reagents

5.2.2. Procedure

6. Summary and prospects

References.

Chapter Ten: A pipeline for characterization of novel Cas9 orthologs.

Notes:

Includes bibliographical references.

Description based on print version record.

ISBN:

0-12-816761-0