CRISPR Guide RNA Design: Methods and Protocols

ISBN-13: 9781071606896 / Angielski / Miękka / 2021

Fulga, Tudor A.

Opis

This detailed volume focuses on the CRISPR-associated guide RNA and how it can be designed, modified, and validated for a broad repertoire of purposes. Beginning with a section on computational design of target-specific guide RNAs, the book continues by covering chemical modifications to alter guide RNA stability, specificity, and efficiency, as well as to create inducible guide RNAs, append additional functional domains, and express guide RNAs in a conditional manner. It concludes with methods for measuring off-target guide RNA activity. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and essential, CRISPR Guide RNA Design: Methods and Protocols provides a comprehensive pipeline for guide RNA design and aims to be an invaluable resource in applying this powerful technology to basic research and therapeutic applications.

Spis treści

Part I: In Silico Design and Optimization of Guide RNA Sequences

 

1. Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments

            Florian Heigwer and Michael Boutros

 

2. Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer

            Gue-Ho Hwang, Jin-Soo Kim, and Sangsu Bae

 

Part II: Chemically-Modified Guide RNAs

 

3. Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells

            Jenny Shapiro, Adi Tovin, Ortal Iancu, Daniel Allen, and Ayal Hendel

 

4. Gene Disruption Using Chemically-Modified CRISPR-Cpf1 RNA

            Moira A. McMahon and Meghdad Rahdar

 

5. ‘Split-and-Click’ sgRNA

            Lapatrada Taemaitree, Arun Shivalingam, Afaf H. El-Sagheer, and Tom Brown

 

6. Chimeric DNA-RNA Guide RNA Designs

            Shuhan Lu, Ying Zhang, and Hao Yin

 

Part III: Expanding the CRISPR Toolbox 

 

7. Harnessing tRNA for Processing Ability and Promoter Activity

            David J.H.F. Knapp and Tudor A. Fulga

 

8. Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA–ncRNA Expression Constructs

            David M. Shechner

 

9. Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs

            Quentin R.V. Ferry

 

10. Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a

            Ha Rim Shin, Jiyeon Kweon, and Yongsub Kim

 

Part IV: Characterization of CRISPR Efficacy and Specificity

 

11. Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells

            Christopher R. Cromwell, Juan Jovel, and Basil P. Hubbard

 

12. In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems

            Christopher R. Cromwell and Basil P. Hubbard

 

13. Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq

            Daesik Kim and Jin-Soo Kim

 

14. Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector

            Xiaoling Wang, Youjun Wu, and Jiing-Kuan Yee

 

15. Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method

            Roberto Ballarino, Britta A.M. Bouwman, and Nicola Crosetto