CRISPR – Cas
A LABORATORY MANUAL
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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
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John Inglis
Richard Sever
Maria Smit
Jan Argentine
Maryliz Dickerson
Carol Brown
Joanne McFadden
Denise Weiss
Wayne Manos
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 sequences. The image depicts the process of capturing foreign DNA, catalyzed by the proteins Cas1 and Cas2, in preparation for integration 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
10 9 8 7 6 5 4 3 2 1
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Readers should please consult individual manufacturers and other resources for current and specific product information.
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Contents
Preface
CHAPTER
1
ix
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
CHAPTER
7
CRISPR – Cas9 Genome Engineering in Saccharomyces cerevisiae Cells
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)
Benjamin E. Housden and Norbert Perrimon
98
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
Dan Du and Lei S. Qi
159
viii / Contents
PROTOCOL
1
CRISPR Technology for Genome Activation and Repression in Mammalian Cells
163
Dan Du and Lei S. Qi
APPENDIX
INDEX
General Safety and Hazardous Material Information
173
179
Preface
Genomes encode the rules for life-forms. Differences in genomes underlie most organismal diversity, 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 comprehensive 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 interspaced 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 incorporating fragments of invading phage and plasmid DNA into CRISPR loci and using the corresponding CRISPR RNAs (crRNAs) to guide the degradation of homologous sequences. Each
CRISPR locus encodes acquired “spacers” that are typically separated by repeat sequences. Transcription 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 understanding 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 methodology, 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 computational 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 genomeediting 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 pluripotent 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 biological 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 performed, 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 property 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.