CRISPR Guide RNA Design: Methods and Protocols

✍ Scribed by Tudor A. Fulga, David J. H. F. Knapp, Quentin R. V. Ferry

• Publisher: Springer US;Humana
• Year: 2021
• Tongue: English
• Leaves: 287
• Series: Methods in Molecular Biology 2162
• Edition: 1st ed.
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Front Matter ....Pages i-x
Front Matter ....Pages 1-1
Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments (Florian Heigwer, Michael Boutros)....Pages 3-22
Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer (Gue-Ho Hwang, Jin-Soo Kim, Sangsu Bae)....Pages 23-33
Front Matter ....Pages 35-35
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, Ayal Hendel)....Pages 37-48
Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA (Moira A. McMahon, Meghdad Rahdar)....Pages 49-60
“Split-and-Click” sgRNA (Lapatrada Taemaitree, Arun Shivalingam, Afaf H. El-Sagheer, Tom Brown)....Pages 61-78
Chimeric DNA–RNA Guide RNA Designs (Shuhan Lu, Ying Zhang, Hao Yin)....Pages 79-85
Front Matter ....Pages 87-87
Harnessing tRNA for Processing Ability and Promoter Activity (David J. H. F. Knapp, Tudor A. Fulga)....Pages 89-114
Targeting Noncoding RNA Domains to Genomic Loci with CRISPR-Display: Guidelines for Designing, Building, and Testing sgRNA–ncRNA Expression Constructs (David M. Shechner)....Pages 115-152
Controlling the Activity of CRISPR Transcriptional Regulators with Inducible sgRNAs (Quentin R. V. Ferry, Tudor A. Fulga)....Pages 153-184
Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a (Ha Rim Shin, Jiyeon Kweon, Yongsub Kim)....Pages 185-193
Front Matter ....Pages 195-195
Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells (Christopher R. Cromwell, Juan Jovel, Basil P. Hubbard)....Pages 197-213
In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems (Christopher R. Cromwell, Basil P. Hubbard)....Pages 215-232
Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq (Daesik Kim, Jin-Soo Kim)....Pages 233-242
Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector (Xiaoling Wang, Youjun Wu, Jiing-Kuan Yee)....Pages 243-260
Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method (Roberto Ballarino, Britta A. M. Bouwman, Nicola Crosetto)....Pages 261-281
Back Matter ....Pages 283-286

✦ Subjects

Life Sciences; Genetics and Genomics; Biomedical Engineering