The Use of CRISPR/cas9, ZFNs, TALENs in Generating Site-Specific Genome Alterations

1st Edition, Volume 546 - November 4, 2014
Editors: Jennifer A. Doudna, Erik J. Sontheimer
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 1 8 5 - 0
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 0 1 3 3 4 - 2

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers recent resear… Read more

The Use of CRISPR/cas9, ZFNs, TALENs in Generating Site-Specific Genome Alterations

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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers recent research and methods development for changing the DNA sequence within the genomes of cells and organisms. Focusing on enzymes that generate double-strand breaks in DNA, the chapters describe use of molecular tools to introduce or delete genetic information at specific sites in the genomes of animal, plant and bacterial cells.

Preface
Chapter One: In Vitro Enzymology of Cas9
- Abstract
- 1 Introduction
- 2 Expression and Purification of Cas9
- 3 Preparation of Guide RNAs
- 4 Endonuclease Cleavage Assays
- 5 Concluding Remarks
- Acknowledgments
Chapter Two: Targeted Genome Editing in Human Cells Using CRISPR/Cas Nucleases and Truncated Guide RNAs
- Abstract
- 1 Introduction
- 2 Methods
- Conflict of Interest
Chapter Three: Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes
- Abstract
- 1 Introduction
- 2 Methods
- 3 Conclusion
- Acknowledgments
Chapter Four: Genome Engineering with Custom Recombinases
- Abstract
- 1 Introduction
- 2 Target Identification
- 3 Recombinase Construction
- 4 Measurements of Recombinase Activity
- 5 Site-Specific Integration
- 6 Conclusions
- Acknowledgments
Chapter Five: Genome Engineering in Human Cells
- Abstract
- 1 Introduction
- 2 Structure of the Human Genome
- 3 Scope of Human Gene Editing Using Programmable Nucleases
- 4 Programmable Nucleases Used for Genome Editing in Human Cells
- 5 Correction of Human Genetic Diseases Using Programmable Nucleases
- 6 Treatment of Human Nongenetic Diseases Using Programmable Nucleases
- 7 Genome Engineering in Human Pluripotent Stem Cells
- 8 Delivery of Programmable Nucleases to Human Cells
- 9 Nickases for Modifying the Human Genome
- 10 Enrichment of Gene-Edited Human Cells
- 11 Conclusion
- Acknowledgments
Chapter Six: Genome Editing in Human Stem Cells
- Abstract
- 1 Introduction
- 2 Gene Targeting Strategies
- 3 Choice of Nuclease Targeting Sites
- 4 Experimental Procedures
- 5 Alternative Approaches
Chapter Seven: Tagging Endogenous Loci for Live-Cell Fluorescence Imaging and Molecule Counting Using ZFNs, TALENs, and Cas9
- Abstract
- 1 Introduction
- 2 Methods
- 3 Tagging/Editing Limitations
- 4 Perspectives
- Acknowledgments
Chapter Eight: Genome Editing Using Cas9 Nickases
- Abstract
- 1 Introduction
- 2 Target Selection
- 3 Plasmid sgRNA Construction
- 4 Validation of sgRNAs in Cell Lines
- 5 Cell Harvest and DNA Extraction
- 6 SURVEYOR Indel Analysis
- 7 HDR and Non-HDR Insertion Using Cas9n
- 8 Analysis of HDR and Insertion Events
- 9 Troubleshooting
- Acknowledgments
Chapter Nine: Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and Cas9 Nucleases
- Abstract
- 1 Introduction
- 2 Cloning the Nickase and Catalytically Dead Variants of Cas9
- 3 Selection of the Target Site and Cloning of sgRNA Constructs
- 4 Cell Transfection and FACS Analysis
- 5 Materials
- 6 Summary
Chapter Ten: Adapting CRISPR/Cas9 for Functional Genomics Screens
- Abstract
- 1 Introduction
- 2 Altering the Vector Design for High-Throughput Screens
- 3 Construction of sgRNA Libraries
- 4 Retroviral Transduction of the Guide Library
- 5 Notes on Screening Design Parameters
- 6 Decoding “Hits” from Positive Selection Screens Involving sgRNA Library Pools
- 7 Conclusion
Chapter Eleven: The iCRISPR Platform for Rapid Genome Editing in Human Pluripotent Stem Cells
- Abstract
- 1 Introduction
- 2 Generation of iCas9 hPSCs
- 3 Generation of Knockout hPSCs Using iCRISPR
- 4 Generation of Precise Nucleotide Alterations Using iCRISPR
- 5 Inducible Gene Knockout in hPSCs Using iCRISPR
- 6 Conclusions and Future Directions
- Acknowledgments
Chapter Twelve: Creating Cancer Translocations in Human Cells Using Cas9 DSBs and nCas9 Paired Nicks
- Abstract
- 1 Introduction
- 2 Materials
- 3 Methods to Induce and Detect Cancer Translocations in Human Cells
- 4 Conclusions
- Acknowledgments
Chapter Thirteen: Genome Editing for Human Gene Therapy
- Abstract
- 1 Introduction
- 2 Genome Editing of B2M in Primary Human CD4+ T Cells
- 3 Targeting of CCR5 in Human CD34+ HSPCs Using CRISPR/Cas9
Chapter Fourteen: Generation of Site-Specific Mutations in the Rat Genome Via CRISPR/Cas9
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: In Vitro Transcription of sgRNA Target Oligonucleotides
- 6 Step 2: In Vitro Transcription of Cas9 mRNA
- 7 Step 3: Preparation of Pseudopregnant Female Rats and One-Cell Rat Embryos
- 8 Step 4: Microinjection of One-Cell Embryos and Transplanting the Embryos into Pseudopregnant Rats
- 9 Step 5: Identification of Founder Rats
- 10 Step 6: Production of F₁ Generation Rats
Chapter Fifteen: CRISPR/Cas9-Based Genome Editing in Mice by Single Plasmid Injection
- Abstract
- 1 Introduction
- 2 Design and Construction of CRISPR/Cas9 Plasmids with pX330
- 3 Validation of pX330 In Vitro
- 4 One-Step Generation of Mutant Mice Via Circular Plasmid Injection
- 5 Screening for Targeted Mutation in Mice
- 6 Concluding Remarks
- Acknowledgment
Chapter Sixteen: Imaging Genomic Elements in Living Cells Using CRISPR/Cas9
- Abstract
- 1 Introduction
- 2 Generation of Cell Lines Stably Expressing dCas9-GFP
- 3 Expression of sgRNAs Using Lentiviral Vector
- 4 Labeling of Nonrepetitive Sequences
- 5 Imaging of Genomic Loci Detected by CRISPR
- 6 Summary
- Acknowledgments
Chapter Seventeen: Cas9-Based Genome Editing in Xenopus tropicalis
- Abstract
- 1 Introduction
- 2 Principle
- 3 Protocol
- 4 Discussion
- Acknowledgments
Chapter Eighteen: Cas9-Based Genome Editing in Zebrafish
- Abstract
- 1 Introduction
- 2 Targeted Generation of Indel Mutations
- 3 Other Targeted Genome-Editing Strategies
- 4 Future Directions
- Acknowledgments
Chapter Nineteen: Cas9-Based Genome Editing in Drosophila
- Abstract
- 1 Introduction
- 2 Applications and Design Considerations for CRISPR-Based Genome Editing
- 3 Delivery of CRISPR Components
- 4 Generation of CRISPR Reagents
- 5 Detection of Mutations
- Acknowledgments
Chapter Twenty: Transgene-Free Genome Editing by Germline Injection of CRISPR/Cas RNA
- Abstract
- 1 Theory, Philosophy, and Practical Considerations
- 2 Equipment
- 3 Materials
- 4 Identifying a Target Sequence
- 5 Generating Your sgRNA Construct
- 6 In Vitro Synthesis of sgRNA
- 7 In Vitro Synthesis of hCas9 mRNA
- 8 Injection of sgRNA and mRNA
- 9 Recovery of Mutants Generated Using CRISPR/Cas
Chapter Twenty-One: Cas9-Based Genome Editing in Arabidopsis and Tobacco
- Abstract
- 1 Introduction
- 2 Cas9 and sgRNA expression
- 3 Dual sgRNA-Guided Genome Editing
- 4 Perspectives
- 5 Notes
- Acknowledgments
Chapter Twenty-Two: Multiplex Engineering of Industrial Yeast Genomes Using CRISPRm
- Abstract
- 1 Introduction
- 2 Plasmid Design
- 3 Cas9 Expression
- 4 Guide RNA Expression
- 5 Screening Method
- 6 Concluding Remarks
- Acknowledgments
Chapter Twenty-Three: Protein Engineering of Cas9 for Enhanced Function
- Abstract
- 1 Introduction
- 2 Methods
- 3 Conclusion
Author Index
Subject Index

Jennifer A. Doudna

Jennifer A. Doudna, Ph.D., professor of Molecular and Cell Biology and Chemistry at the University of California, Berkeley and Howard Hughes Medical Institute investigator, has devoted her scientific career to revealing the secret life of RNA. Using the approaches of structural biology and biochemistry, Doudna’s work deciphering the molecular structure of RNA enzymes and other functional RNAs has shown how these seemingly simple molecules can carry out complex functions and can work together with proteins to control the information content of a cell.

Doudna grew up amidst the natural wonders of Hawaii, where she experienced volcanic eruptions, explored remote beaches and honed her body-surfing skills while living in the small town of Hilo on the Big Island. Doudna earned a B.A. in Biochemistry at Pomona College in 1985, where she worked with outstanding chemists Sharon Panasenko and Fred Grieman, and enjoyed the mentorship of many other great professors. She then worked with Jack Szostak at Harvard, completing her Ph.D. in 1989 on the develpoment of a self-replicating RNA based on the activity of a group I self-splicing intron. This work showed how RNA could function as both a template and a catalyst for generating copies of itself, a key propoerty of life. As a Lucille Markey postdoctoral associate with Tom Cech at University of Colorado at Boulder, Doudna began crystallizing catalytic RNA molecules with a goal of determining their three-dimensioanl structures and hence inlocking the key to their biochemical activities. She continued this work as a faculty member at Yale University, where she became a professor in 1994 in the Department of Molecular Biophysics and Biochemistry. In two landmark studies early in her career, Doudna and colleagues solved the crystal structures of two large RNAs – the P4-P6 domain of the Tetrahymena thermophila group I intron ribozyme and the hepatitis delta virus ribozyme. By determining their molecular structures, her work advanced the understanding of RNA’s ability to function as a catalyst in biological systems. In 2002, Doudna moved to the University of California at Berkeley, where her lab began studying the function of small RNAs that control the use of a cell’s genetic information. This led to her work on bacterial immune systems that employ RNA molecules derived from viruses to target and destroy foreign DNA. In collaboration with the lab of Emmanuelle Charpentier, Doudna and postdoctoral associate Martin Jinek discovered the function of an RNA-guided enzyme in the bacterial immune pathway, Cas9, whose ability to cut double-stranded DNA can be programmed by changing the guide RNA sequence. They recognized that such an activity could be employed as a molecular tool for precision genome engineering in various kinds of cells, a discovery that has triggered a revolution in the fields of molecular genetics and genomics.

Doudna’s work has been honored by numerous awards. She received the National Academy of Sciences Award for Initiatives in Research in 1999, and the Alan T. Waterman Award from the NSF in 2000. In 2001 she received the Eli Lilley Award in Biological Chemistry from the American Chemical Society, and in 2013 she was the recipient of the Mildred Cohn Award from ASBMB and the Hans Neurath Award from the Protein Society. She has been a Howard Hughes Medical Institute investigator since 1997 and a member of the National Academy of Sciences since 2002. She was named to the American Academy of Arts and Sciences in 2003 and elected to the Institute of Medicine in 2010. In 2014 she received the Lurie Prize from the Foundation for the NIH.

Affiliations and expertise

Molecular & Cell Biology & Chemistry, University of California, Berkeley; Howard Hughes Medical Institute, USA

Erik J. Sontheimer

Erik J. Sontheimer, Ph.D., is Professor in the RNA Therapeutics Institute and the Program for Molecular Medicine at the University of Massachusetts Medical School. A native of Pittsburgh, he attended the Pennsylvania State University, where he received his B.S. in Molecular and Cell Biology in 1987. He then moved to the Department of Molecular Biophysics and Biochemistry at Yale University where he worked with Joan Steitz, completing his Ph.D. in 1992. His work at Yale revealed a dynamic network of RNA-RNA interactions that help to identify and excise non-coding “intron” sequences from eukaryotic messenger RNA precursors. He then did postdoctoral work with Joe Piccirilli in the Department of Biochemistry and Molecular Biology at the University of Chicago, where he was a Fellow of the Jane Coffin Childs Memorial Fund. While at Chicago he provided the first glimpses of the chemical strategies that eukaryotic cells use to catalyze the reactions that remove introns from pre-messenger RNAs.

In 1999, Sontheimer joined the faculty in the Department of Molecular Biosciences at Northwestern University in Evanston, Illinois, where he continued his work on the mechanisms of pre-mRNA splicing. He also turned his attention to small RNA-based gene regulation, and his laboratory made fundamental contributions to the understanding of RNA interference pathways. In 2008 his laboratory also began working on genetic interference mechanisms in pathogenic bacteria. Among other advances, they provided the first demonstration that small RNAs known as CRISPR RNAs target DNA molecules directly, paving the way for the development of RNA-guided genome engineering applications. While at Northwestern, he received a CAREER Award from the National Science Foundation, a New Investigator Award in the Basic Pharmacological Sciences from the Burroughs Wellcome Fund, a Basil O’Conner Award from the March of Dimes, a Scholar Award from the American Cancer Society, a Distinguished Teaching Award from the Weinberg College of Arts and Sciences, and the 2008 Nestle Award from the American Society for Microbiology. In the summer of 2014 he moved to the RNA Therapeutics Institute at the University of Massachusetts Medical School in Worcester, Massachusetts, where he is continuing his research on the fundamental roles of RNA molecules in gene expression, and on the uses of RNA molecules in biomedical research and the treatment of human disease.

Affiliations and expertise

Molecular Biosciences, Northwestern University, USA

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