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WEBSITE www.cshprotocols.org CRISPR – Cas A LABORATORY MANUAL
EDITED BY
Jennifer Doudna University of California, Berkeley
Prashant Mali University of California, San Diego
COLD S PRING H ARBOR L ABORATORY P RESS
C o l d S p r i n g H a r b o r , N ew Yo r k † w w w . c s h l p r e s s . o r g CRISPR–Cas A LABORATORY MANUAL
All rights reserved
# 2016 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Printed in the United States of America
Publisher John Inglis
Acquisition Editor Richard Sever Managing Editor Maria Smit Director of Editorial Services Jan Argentine Project Manager Maryliz Dickerson Permissions Coordinator Carol Brown Production Editor Joanne McFadden Production Manager Denise Weiss Director of Product Development & Marketing Wayne Manos Cover Designer Denise Weiss
Cover art: CRISPR – Cas adaptive immunity in bacteria and archaea relies on the capture of invading foreign DNA and its subsequent integration into the CRISPR locus to elicit memory of the infection. The RNA products of newly captured DNA, called spacers, are used by Cas proteins, such as Cas9, as guides for destruction of complementary foreign DNA sequen- ces. The image depicts the process of capturing foreign DNA, catalyzed by the proteins Cas1 and Cas2, in preparation for inte- gration into the host’s CRISPR locus. Credit: Megan Riel-Mehan and Graham Johnson (UCSF).
Library of Congress Cataloging-in-Publication Data
Names: Doudna, Jennifer A., editor. | Mali, Prashant, editor. Title: CRISPR-Cas: a laboratory manual/edited by Jennifer Doudna, Prashant Mali. Description: Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, [2016] | Includes bibliographical references and index. Identifiers: LCCN 2015050261 | ISBN 9781621821304 (hardcover: alk. paper) | ISBN 9781621821311 ( pbk. : alk. paper) Subjects: | MESH: CRISPR-Cas Systems | CRISPR-Associated Proteins | Genetic Techniques | Laboratory Manuals Classification: LCC QP623 | NLM QU 25 | DDC 572.8/8078- -dc23 LC record available at http://lccn.loc.gov/2015050261
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Students and researchers using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no repre- sentations or warranties with respect to the material set forth in this manual and has no liability in connection with the use of these materials. All registered trademarks, trade names, and brand names mentioned in this book are the property of the respective owners. Readers should please consult individual manufacturers and other resources for current and specific product information. With the exception of those suppliers listed in the text with their addresses, all suppliers mentioned in this manual can be found on the BioSupplyNet website at www.biosupplynet.com. All World Wide Web addresses are accurate to the best of our knowledge at the time of printing. Procedures for the humane treatment of animals must be observed at all times. Check with the local animal facility for guidelines. Certain experimental procedures in this manual may be the subject of national or local legislation or agency restrictions. Users of this manual are responsible for obtaining the relevant permissions, certificates, or licenses in these cases. Neither the authors of this manual nor Cold Spring Harbor Laboratory assume any responsibility for failure of a user to do so. The materials and methods in this manual may infringe the patent and proprietary rights of other individuals, companies, or organiza- tions. Users of this manual are responsible for obtaining any licenses necessary to use such materials and to practice such methods. COLD SPRING HARBOR LABORATORY MAKES NO WARRANTY OR REPRESENTATION THAT USE OF THE INFORMATION IN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR OTHER PROPRIETARY RIGHT. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Cold Spring Harbor Laboratory Press, provided that the appropriate fee is paid directly to the Copyright Clearance Center (CCC). Write or call CCC at 222 Rosewood Drive, Danvers, MA 01923 (978-750-8400) for information about fees and regulations. Prior to photocopying items for educa- tional classroom use, contact CCC at the above address. Additional information on CCC can be obtained at CCC Online at www.copyright.com. For a complete catalog of all Cold Spring Harbor Laboratory Press publications, visit our website at www.cshlpress.org. Contents
Preface ix
CHAPTER 1 Overview of CRISPR – Cas9 Biology
INTRODUCTION Overview of CRISPR – Cas9 Biology 1 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 17 Alexandra E. Briner and Rudolphe Barrangou
PROTOCOL 1 Prediction and Validation of Native and Engineered Cas9 Guide Sequences 24 Alexandra E. Briner, Emily D. Henriksen, and Rodolphe Barrangou
CHAPTER 3 Characterization of Cas9 – Guide RNA Orthologs
INTRODUCTION Characterization of Cas9 – Guide RNA Orthologs 31 Jonathan L. Braff, Stephanie J. Yaung, Kevin M. Esvelt, and George M. Church
PROTOCOL 1 Characterizing Cas9 Protospacer-Adjacent Motifs with High-Throughput Sequencing of Library Depletion Experiments 35 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 39 Tim Wang, Eric S. Lander, and David M. Sabatini
PROTOCOLS 1 Single Guide RNA Library Design and Construction 43 Tim Wang, Eric S. Lander, and David M. Sabatini
v vi / Contents
2 Viral Packaging and Cell Culture for CRISPR-Based Screens 49
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 1 Adeno-Associated Virus – Mediated Delivery of CRISPR – Cas Systems for Genome Engineering in Mammalian Cells 57 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 69 Yuichiro Miyaoka, Amanda H. Chan, and Bruce R. Conklin
PROTOCOL 1 Using Digital Polymerase Chain Reaction to Detect Single-Nucleotide Substitutions Induced by Genome Editing 73 Yuichiro Miyaoka, Amanda H. Chan, and Bruce R. Conklin
PROTOCOL 1 CRISPR – Cas9 Genome Engineering in Saccharomyces cerevisiae Cells 79 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 89 Benjamin E. Housden and Norbert Perrimon
PROTOCOLS 1 Design and Generation of Donor Constructs for Genome Engineering in Drosophila 93 Benjamin E. Housden and Norbert Perrimon 2 Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA) 98 Benjamin E. Housden and Norbert Perrimon Contents / vii
3 Design and Generation of Drosophila Single Guide RNA Expression Constructs 106 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 113 Charles E. Vejnar, Miguel Moreno-Mateos, Daniel Cifuentes, Ariel A. Bazzini, and Antonio J. Giraldez
PROTOCOL 1 Optimized CRISPR – Cas9 System for Genome Editing in Zebrafish 117 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 133 Adam Williams, Jorge Henao-Mejia, and Richard A. Flavell
PROTOCOL 1 Generation of Genetically Modified Mice Using the CRISPR –Cas9 Genome-Editing System 139 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 149 Cory Smith, Zhaohui Ye, and Linzhao Cheng
PROTOCOL 1 A Method for Genome Editing in Human Pluripotent Stem Cells 153 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 159 Dan Du and Lei S. Qi viii / Contents
PROTOCOL 1 CRISPR Technology for Genome Activation and Repression in Mammalian Cells 163 Dan Du and Lei S. Qi
APPENDIX General Safety and Hazardous Material Information 173
INDEX 179 Preface
Genomes encode the rules for life-forms. Differences in genomes underlie most organismal diver- sity, and aberrations in genomes underlie many disease states. With the rapid advances in DNA sequencing, we now have near-complete genomes for a range of organisms and a fairly comprehen- sive catalog of human germline and somatic variants, as well as rich annotations of functional genomic elements. The next frontier in the field is to obtain a complete functional annotation of genetic variants and genomic elements at the cellular and organismal levels. Such an understanding, especially in the human context, will not only pave the way for a deeper understanding of the genomic code but will also power therapeutic interventions directed at both effecting cures and eventually also engineering disease resistance. Consequently as we move from reading genomes to interpreting genomes and ultimately engineering genomes, technologies to directly and precisely perturb genomic elements and combinations thereof will be a most critical toolset in these basic science cum engineering endeavors. In this regard, the recent advent of RNA-guided effectors derived from clustered regularly inter- spaced short palindromic repeats (CRISPR)–CRISPR-associated systems (Cas) has dramatically transformed our ability to engineer the genomes of diverse organisms. As unique factors capable of colocalizing RNA, DNA, and protein, tools and techniques based on CRISPR–Cas are paving the way for unprecedented control over cellular organization, regulation, and behavior. Notably, CRISPR–Cas systems evolved as adaptive immune defenses of bacteria and archaea and use short RNA to direct degradation of foreign nucleic acids. They provide immunity by incor- porating fragments of invading phage and plasmid DNA into CRISPR loci and using the corre- sponding CRISPR RNAs (crRNAs) to guide the degradation of homologous sequences. Each CRISPR locus encodes acquired “spacers” that are typically separated by repeat sequences. Tran- scription of the locus yields a pre-crRNA, which is processed to yield crRNAs that guide effector nuclease complexes to disrupt sequences complementary to the spacer. CRISPR systems are thus readily retargeted by expressing or delivering appropriate crRNAs, and progressive mechanistic insights into these fundamental processes thus paved the way for their recent engineering into a range of prokaryotic and eukaryotic organisms. In considering the developments in this rapidly evolving field and its applications for under- standing basic biology and engineering of new therapeutic paradigms, our goal in developing this book was to highlight the major advances that have been made that have led to the current state of research, while also providing a guide for implementation of these approaches. As such, the book is divided into multiple parts and, focusing specifically on the CRISPR–Cas9 targeting meth- odology, it details protocols for applications in a range of species and in ex vivo cum in vivo genome targeting scenarios. We begin with an overview of CRISPR–Cas9 biology, followed by computa- tional and experimental protocols for prediction and validation of native and engineered Cas9 orthologs and guide sequences. Toward harnessing the massively multiplexable and scalable genome engineering enabled by this platform, we next detail protocols for constructing CRISPR libraries for effecting large-scale genetic screens in human cell lines. Given the impending applications of CRISPR–Cas in engineering therapeutics, protocols on establishing an adeno-associated virus– based delivery system into cells and mice are provided next. High-resolution assaying of genomic changes induced by this platform are critical for effectively implementing this approach, and thus we also detail highly sensitive polymerase chain reaction (PCR)-based assays to quantify genome- editing events. We follow this with a collection of protocols for precision genome engineering in a range of organisms including yeast, fruit flies, zebrafish, and mice, as well as human induced plu- ripotent stem cells. We conclude by detailing protocols to enable targeted genome regulation using
ix x / Preface
the CRISPR–Cas9 platform. These chapters provide a comprehensive, in-depth overview of the experimental procedures prevalent in the field. Looking forward, we anticipate the versatility and ease of use afforded by CRISPR–Cas effectors, coupled with their singular ability to bring together RNA, DNA, and protein in a fully programmable fashion, to form the basis of a progressively expanding experimental toolset for the perturbation, regulation, and monitoring of complex biolog- ical systems. We would like to thank the many scientists who have contributed to this book. We are very grateful for their enthusiasm, hard work, and attention to detail in preparing this book, which can serve as a broad resource for technicians, graduate students, postdocs, and any investigator engaged in genetic studies. Special thanks also go to Maryliz Dickerson at Cold Spring Harbor Laboratory Press for helping make this book a reality. Jennifer Doudna Prashant Mali
General Safety and Hazardous Material Information
This manual should be used by laboratory personnel with experience in laboratory and chemical safety or students under the supervision of such trained personnel. The procedures, chemicals, and equipment referenced in this manual are hazardous and can cause serious injury unless per- formed, handled, and used with care and in a manner consistent with safe laboratory practices. Students and researchers using the procedures in this manual do so at their own risk. It is essential for your safety that you consult the appropriate Material Safety Data Sheets, the manufacturers’ manuals accompanying equipment, and your institution’s Environmental Health and Safety Office, as well as the General Safety and Disposal Cautions in the Appendix for proper handling of hazardous materials in this manual. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this manual and has no liability in connection with the use of these materials. All registered trademarks, trade names, and brand names mentioned in this book are the prop- erty of the respective owners. Readers should please consult individual manufacturers and other resources for current and specific product information. Appropriate sources for obtaining safety information and general guidelines for laboratory safety are provided in the General Safety and Hazardous Material Information Appendix.
