Main description:

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.

Contents:

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