CRISPR-Cas Enzymes. (2019)

• Record Type: Book
• Further Information: Note: Edited by Scoll Bailey.
• Editors: Bailey, Scott
• 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 RNASeq5. 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. Introduction; 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 analysis3. Conclusion; Acknowledgments; References; Further reading; Chapter Three: Sortase-mediated fluorescent labeling of CRISPR complexes; 1. Introduction; 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 Cas22.4.2. 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; Acknowledgments; Funding; References; Chapter Four: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems; 1. Introduction 2. Fluorescence-based strategies for measuring CRISPR interference2.1. 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
• Publisher Details: Cambridge, MA : Academic Press
• Publication Date: 2019
• Extent: 1 online resource
• Subjects: 572.86; Genetic engineering; CRISPR (Genetics); Genomics; Endonucleases; Restriction enzymes, DNA; SCIENCE -- Chemistry -- Industrial & Technical; TECHNOLOGY & ENGINEERING -- Chemical & Biochemical; Electronic books
• Languages: English
• ISBNs: 9780128167618; 0128167610; 9780128167601; 0128167602
• Notes: Note: Includes bibliographical references at the end of each chapter and indexes.; Note: Online resource; title from PDF title page (EBSCO, viewed January 31, 2019).; Note: Vendor-supplied metadata.
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