CRISPR Gene Editing Methods and Protocols

About this book

This detailed volume guides readers through strategic planning and user-friendly guidelines in order to select the most suitable CRISPR-Cas system and target sites with high activity and specificity. Methods covering CRISPR gRNA design, CRISPR delivery, CRISPR activity quantification (indel quantification), and examples of applying CRISPR gene editing in human pluripotent stem cells, primary cells, gene therapy, and genetic screening are included. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and invaluable, CRISPR Gene Editing: Methods and Protocols will assist undergraduates, graduates, and researchers with detailed guidelines and methods for the vitally important CRISPR gene editing field.

Contents

Part I: Methods for CRISPR-gRNA Design and Quantification of Activity
1. CRISPR-gRNA Design / Maria Pallares Masmitja, Nastassia Johanna Knoedlseder, and Marc Guell
2. Tracking CRISPR's Footprints / Lin Lin and Yonglun Luo
3. Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER / Eva Karina Brinkman and Bas van Steensel
4. Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA / Saskia Koenig, Yang Zhang, Hans Heugh Wandall, Claudio Mussolino, and Eric Paul Bennett
5. Functional Evaluation of CRISPR Activity by the Dual-Fluorescent Surrogate System: C-Check / Lin Lin and Yonglun Luo

Part II: Methods for CRISPR Delivery
6. CRISPR-Cas9 Delivery by Artificial Virus (RRPHC) / Suleixin Yang, Qinjie Wu, Yuquan Wei, and Changyang Gong
7. Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery / Laura Barrett Ryo, Emil Aagaard Thomsen, and Jacob Giehm Mikkelsen
8. Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors / Julia Fakhiri, Manuela Nickl, and Dirk Grimm
9. Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs / Anders Laustsen and Rasmus O. Bak

Part III: CRISPR Gene Editing in Human iPSCs
10. Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 / Saniye Yumlu, Sanum Bashir, Jurgen Stumm, and Ralf Kuhn
11. Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes / Michaela Bruntraeger, Meg Byrne, Kathleen Long, and Andrew R. Bassett
12. Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9 / Kirsten E. Snijders, James D. Cooper, Ludovic Vallier, and Alessandro Bertero

Part IV: CRISPR Gene Editing in Other Cell Types
13. CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells / Daria Sergeeva, Jose Manuel Camacho-Zaragoza, Jae Seong Lee, and Helene Faustrup Kildegaard
14. Rapid and Efficient Gene Deletion by CRISPR/Cas9 / Signe Neldeborg, Lin Lin, Magnus Stougaard, and Yonglun Luo
15. Genome Editing in Mice / Lisbeth Ahm Hansen and Ernst-Martin Fuchtbauer
16. CRISPR/Cas9-Mediated Gene Tagging: A Step-by-Step Protocol / Xi Xiang, Conghui Li, Xi Chen, Hongwei Dou, Yong Li, Xiuqing Zhang, and Yonglun Luo
17. Gene Editing in Primary Cells of Cattle and Pig / Petra Vochozkova, Kilian Simmet, Eva-Maria Jemiller, Annegret Wunsch, and Nikolai Klymiuk

Part V: CRISPR Gene Therapy and Screening
18. Toward In Vivo Gene Therapy Using CRISPR / Kristian Alsbjerg Skipper and Jacob Giehm Mikkelsen
19. CRISPR Gene Therapy of the Eye: Targeted Knockout of Vegfa in Mouse Retina by Lentiviral Delivery / Andreas Holmgaard, Sidsel Alsing, Anne Louise Askou, and Thomas J. Corydon
20. In Vivo Editing of the Adult Mouse Liver Using CRISPR/Cas9 and Hydrodynamic Tail Vein Injection / Francesco Niola, Frederik Dagnaes-Hansen, and Morten Froedin
21. CRISPR-Based Lentiviral Knockout Libraries for Functional Genomic Screening and Identification of Phenotype-Related Genes / Emil Aagaard Thomsen and Jacob Giehm Mikkelsen

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