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The development of CRISPR-Cas technology is revolutionizing biology. Based on machinery bacteria use to target foreign nucleic acids, these powerful techniques allow investigators to edit nucleic acids and modulate gene expression more rapidly and accurately than ever before.
Featuring contributions from leading figures in the CRISPR-Cas field, this laboratory manual presents a state-of-the-art guide to the technology. It includes step-by-step protocols for applying CRISPR-Cas-based techniques in various systems, including yeast, zebrafish, Drosophila, mice, and cultured cells (e.g., human pluripotent stem cells). The contributors cover web-based tools and approaches for designing guide RNAs that precisely target genes of interest, methods for preparing and delivering CRISPR-Cas reagents into cells, and ways to screen for cells that harbor the desired genetic changes. Strategies for optimizing CRISPR-Cas in each system – especially for minimizing off-target effects – are also provided.
Authors also describe other applications of the CRISPR-Cas system, including its use for regulating genome activation and repression, and discuss the development of next-generation CRISPR-Cas tools. CRISPR-Cas: A Laboratory Manual is thus an essential laboratory resource for all cell, molecular, and developmental biologists, as well as biochemists, geneticists, and all who seek to expand their biotechnology toolkits.
Preface
CHAPTER 1 Overview of CRISPR-Cas9 Biology
INTRODUCTION
Overview of CRISPR-Cas9 Biology
Hannah K. Ratner, Timothy R. Sampson, and David S. Weiss
CHAPTER 2 Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems
INTRODUCTION
Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems
Alexandra E. Briner and Rudolphe Barrangou
PROTOCOL
Prediction and Validation of Native and Engineered Cas9 Guide Sequences
Alexandra E. Briner, Emily D. Henriksen, and Rodolphe Barrangou
CHAPTER 3 Characterization of Cas9–Guide RNA Orthologs
INTRODUCTION
Characterization of Cas9–Guide RNA Orthologs
Jonathan L. Braff, Stephanie J. Yaung, Kevin M. Esvelt, and George M. Church
PROTOCOL
Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments
Jonathan L. Braff, Stephanie J. Yaung, Kevin M. Esvelt, and George M. Church
CHAPTER 4 Large-Scale Single Guide RNA Library Construction and Use for CRISPR-Cas9-Based Genetic Screens
INTRODUCTION
Large-Scale Single Guide RNA Library Construction and Use for CRISPR-Cas9-Based Genetic Screens
Tim Wang, Eric S. Lander, and David M. Sabatini
PROTOCOLS
Single Guide RNA Library Design and Construction
Tim Wang, Eric S. Lander, and David M. Sabatini
Viral Packaging and Cell Culture for CRISPR-Based Screens
Tim Wang, Eric S. Lander, and David M. Sabatini
CHAPTER 5 Adeno-Associated Virus–Mediated Delivery of CRISPR-Cas Systems for Genome Engineering in Mammalian Cells
PROTOCOL
Adeno-Associated Virus–Mediated Delivery of CRISPR-Cas Systems for Genome Engineering in Mammalian Cells
Thomas Gaj and David V. Schaffer
CHAPTER 6 Detecting Single-Nucleotide Substitutions Induced by Genome Editing
INTRODUCTION
Detecting Single-Nucleotide Substitutions Induced by Genome Editing
Yuichiro Miyaoka, Amanda H. Chan, and Bruce R. Conklin
PROTOCOL
Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing
Yuichiro Miyaoka, Amanda H. Chan, and Bruce R. Conklin
CHAPTER 7 CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells
PROTOCOL
CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells
Owen W. Ryan, Snigdha Poddar, and Jamie H.D. Cate
CHAPTER 8 Cas9-Mediated Genome Engineering in Drosophila melanogaster
INTRODUCTION
Cas9-Mediated Genome Engineering in Drosophila melanogaster
Benjamin E. Housden and Norbert Perrimon
PROTOCOLS
Design and Generation of Donor Constructs for Genome Engineering in Drosophila
Benjamin E. Housden and Norbert Perrimon
Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA)
Benjamin E. Housden and Norbert Perrimon
Design and Generation of Drosophila Single Guide RNA Expression Constructs
Benjamin E. Housden, Yanhui Hu, and Norbert Perrimon
CHAPTER 9 Optimization Strategies for the CRISPR-Cas9 Genome-Editing System
INTRODUCTION
Optimization Strategies for the CRISPR-Cas9 Genome-Editing System
Charles E. Vejnar, Miguel Moreno-Mateos, Daniel Cifuentes, Ariel A. Bazzini, and Antonio J. Giraldez
PROTOCOL
Optimized CRISPR-Cas9 System for Genome Editing in Zebrafish
Charles E. Vejnar, Miguel Moreno-Mateos, Daniel Cifuentes, Ariel A. Bazzini, and Antonio J. Giraldez
CHAPTER 10 Editing the Mouse Genome Using the CRISPR-Cas9 System
INTRODUCTION
Editing the Mouse Genome Using the CRISPR-Cas9 System
Adam Williams, Jorge Henao-Mejia, and Richard A. Flavell
PROTOCOL
Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System
Jorge Henao-Mejia, Adam Williams, Anthony Rongvaux, Judith Stein, Cynthia Hughes, and Richard A. Flavell
CHAPTER 11 Genome Editing in Human Pluripotent Stem Cells
INTRODUCTION
Genome Editing in Human Pluripotent Stem Cells
Cory Smith, Zhaohui Ye, and Linzhao Cheng
PROTOCOL
A Method for Genome Editing in Human Pluripotent Stem Cells
Cory Smith, Zhaohui Ye, and Linzhao Cheng
CHAPTER 12 An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells
INTRODUCTION
An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells
Dan Du and Lei S. Qi
PROTOCOL
CRISPR Technology for Genome Activation and Repression in Mammalian Cells
Dan Du and Lei S. Qi
APPENDIX General Safety and Hazardous Material Information
INDEX
