CRISPR-Cas Enzymes /

• Άλλοι συγγραφείς: Bailey, Scott (Επιμελητής έκδοσης)
• Μορφή: Ηλ. βιβλίο
• Γλώσσα: English
• Έκδοση: Cambridge, MA : Academic Press, 2019.
• Έκδοση: First edition.
• Σειρά: Methods in enzymology ; v. 616.
• Θέματα: Genetic engineering.; CRISPR (Genetics); Genomics.; Endonucleases.; Restriction enzymes, DNA.; CRISPR-Associated Protein 9.; CRISPR-Cas Systems.; Clustered Regularly Interspaced Short Palindromic Repeats.; Gene Editing > methods.; SCIENCE > Chemistry > Industrial & Technical.; TECHNOLOGY & ENGINEERING > Chemical & Biochemical.; Electronic books.

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 a...

Πίνακας περιεχομένων:

• 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