CRISPR E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Serie: ASM
- Sprache: Englisch
“An excellent compendium of all things CRISPR from some of the leading minds in the field. With thorough coverage from every angle and beautifully detailed illustrations, this book is not to be missed!”
Jennifer A. Doudna,
Professor of Chemistry, Biochemistry & Molecular Biology, UC Berkeley; Founder, Innovative Genomics Institute; Nobel Laureate and coinventor of CRISPR technology
“This journey through CRISPR biology and several of its breakthrough applications offers an exciting glimpse into one of the most beautiful and compelling fields in the life sciences.”
David R. Liu,
Director of the Merkin Institute at the Broad Institute of MIT and Harvard; Professor of Chemistry and Chemical Biology at Harvard University; coinventor of base editing and prime editing
“A must read! The CRISPR topics, written by world-leading experts, span from the fascinating mechanistic underpinnings to the ingenious applications. One can read from start to finish or pick and choose themes. Either way, the book delivers utterly enjoyable learning!”
Bonnie Bassler,
Squibb Professor and Chair, Princeton University Department of Molecular Biology; Howard Hughes Medical Institute Investigator
CRISPR-Cas systems have revolutionized the science of gene editing and their possible applications continue to expand, from basic research to potentially groundbreaking medical and commercial uses.
Led by a distinguished team of editors, CRISPR: Biology and Applications explores the subject matter needed to delve into this fascinating area. Topics covered include:
- Classification and molecular mechanisms of CRISPR-Cas systems
- CRISPR-Cas evolution, regulation, expression, and function
- Uses for gene editing and modulation of gene expression
- CRISPR-based antimicrobials and phage resistance for medical and industrial purposes
Written by internationally renowned authors, CRISPR: Biology and Applications serves as both an introductory guide for those new to the field and an authoritative reference for seasoned researchers whose work touches this evolving and headline- making science.
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Seitenzahl: 893
Veröffentlichungsjahr: 2022
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Table of Contents
Cover
Title Page
Copyright
Contributors
Preface
Section I: Diversity of CRISPR‐Cas Systems
Chapter 1: CRISPR‐Cas Systems: Core Features and Common Mechanisms
Introduction
The CRISPR Array
Spacer Acquisition
CRISPR Targets
RNA‐Guided Nucleases
Evasion of CRISPR Immunity
Conclusions
References
Chapter 2: Evolutionary Classification of CRISPR‐Cas Systems
Introduction
Principles of CRISPR‐Cas System Classification
Classification of CRISPR‐Cas Systems
Distribution of Different Variants of CRISPR‐Cas Systems Among Bacteria and Archaea
A Brief Evolutionary History of CRISPR
Conclusions and Outlook
Acknowledgments
References
Chapter 3: Molecular Mechanisms of Type I CRISPR‐Cas Systems
Introduction
CRISPR‐Cas Mechanism
Spacer Acquisition
Applications
Conclusion and Outlook
References
Chapter 4: Molecular Mechanisms of Type II CRISPR‐Cas Systems
A Brief Overview of Type II CRISPR‐Cas Systems
Discovery of Type II CRISPR‐Cas Systems
The Biology of Type II CRISPR‐Cas Systems
Cas9: Programmable RNA‐Guided DNA Endonuclease
Adaptation of Cas9 Nuclease for
In Vivo
Genome Modification
Mechanism of DNA Cleavage by Cas9 Nuclease
Cas9 Challenges and Limitations
Concluding Remarks
References
Chapter 5: Mechanism of Type III CRISPR‐Cas Immunity
Introduction
Type III CRISPR‐Cas Immunity Requires Target Transcription
crRNA Biogenesis
RNA Recognition
DNA Degradation
Activation of CARF Accessory Proteins by cOA Second Messengers
Inactivation of the Type III CRISPR‐Cas Immune Response
Target Sequence Requirements: The PAM
Target Sequence Requirements: Protospacer Mutations
Robustness of Type III CRISPR‐Cas Immune Response
Spacer Acquisition
The Future of Type III CRISPR‐Cas Research: Genetic Diversity and Accessory Effectors
Acknowledgments
References
Chapter 6: Type V CRISPR‐Cas Systems
Introduction to Type V Systems
Evolution and Classification of Type V Systems
Domain Organization and Structure of Type V Effector Proteins
Spacer Adaptation in Type V Systems
RNA Processing in Type V Systems
CRISPR Interference by Cas12 Effector Proteins
The Transposon‐Associated Type V‐K Systems
Conclusion
References
Chapter 7: CRISPR‐Cas13: Biology, Mechanism, and Applications of RNA‐Guided, RNA‐Targeting CRISPR Systems
Discovery of CRISPR‐Cas13
Diversity in the Cas13 Family
Role of Cas13 in Bacterial Defense
Mechanisms Underlying Cas13‐Mediated Target Cleavage and Pre‐cRNA Processing
Mechanisms Underlying Nonspecific Collateral Activity
Comparison of Cas13 to Type III CRISPR Systems and Other HEPN‐Associated Systems
Roles of Accessory Proteins in Cas13 Interference
Applications of Cas13
Future of Cas13
Acknowledgments
References
Section II: CRISPR‐Cas Biology
Chapter 8: CRISPR‐Cas, Horizontal Gene Transfer, and the Flexible (Pan) Genome
Introduction
Proviruses/Prophages
Natural Transformation
Antibiotic Resistance
Mobility of CRISPR‐Cas Systems
CRISPR‐Cas Systems as Pangenome Editors
Acknowledgments
References
Chapter 9: Evasion Tactics Manifested by Bacteriophages against Bacterial Immunity
Introduction
Restriction‐Modification Systems
CRISPR‐Cas
Future Directions
References
Chapter 10: Regulation of CRISPR‐Cas Expression and Function
Introduction
QS Influences CRISPR‐Cas Immunity
CRISPR‐Cas Repression through H‐NS
CRP Modulates CRISPR‐Cas Immunity
Specialized Sigma Factors Influence CRISPR‐Cas Expression
Stress Response Systems Implicated in CRISPR‐Cas Regulation
Viral Infection Stimulates CRISPR‐Cas Response
Autoregulators and Exclusive CRISPR Regulators
Potential Cooperation between Defense Systems
Future Directions
Acknowledgments
References
Section III CRISPR‐Based Technologies and Applications
Chapter 11: Genome Editing with CRISPR‐Cas Systems
Introduction to Genome Editing
DNA Targeting with CRISPR‐Cas Nucleases
DNA Repair and Genome Editing Outcomes
Applications
Therapeutic Genome Editing
Conclusion and Perspectives
References
Chapter 12: Genetic and Epigenetic Modulation of Gene Expression by CRISPR‐dCas Systems
Introduction
Nuclease‐Deficient CRISPR‐Associated 9 Systems for Programmable Gene Regulation
Repression of Transcription via CRISPR Interference
Activation of Transcription via CRISPR Activation
Improvements of dCas9‐Coupled Activators of Gene Expression
Epigenetic Control of Transcription by dCas9 Effector Fusion Proteins
CRISPR‐Mediated Chromatin Repositioning and Modulation of the Three‐Dimensional Genome: CRISPR‐Genome Organizer and CRISPR‐Engineered Chromatin Organization
Applications of CRISPR‐dCas9‐Mediated Manipulation of Gene Expression for Basic Cell Biology, Regenerative Medicine, and Gene Therapy
Off‐Targeting Issues in dCas9 Fusion‐Mediated Transcriptional Regulation
Future Perspectives
References
Chapter 13: CRISPR Screens
Introduction
Genetic Screens
Overview of CRISPR/Cas9 Screens
Screen Design, Execution, and Analysis
Limitations and Improvements of CRISPR Screens
Expansion of the Scope of CRISPR Screens
Illuminating Bacterial Host‐Pathogen Interactions with CRISPR Screens
Final Thoughts
Acknowledgments
References
Chapter 14: CRISPR‐Based Antimicrobials
Introduction
Mechanisms of CRISPR‐Cas Antimicrobials
Delivery Systems
Considerations
Future Prospects
Acknowledgment
References
Chapter 15: Exploiting CRISPR‐Cas Systems To Provide Phage Resistance in Industrial Bacteria
Introduction
CRISPR‐Mediated Host‐Phage Coevolutionary Dynamics in
Streptococcus thermophilus
Expanding CRISPR Immunity in Industrial Bacteria
Using CRISPR Hypervariability for Genotyping
Applications of CRISPR Immunity
Conclusions and Outlook
Acknowledgments
References
Chapter 16: Recording Biological Information with CRISPR‐Cas Systems
Introduction
DNA Writing with CRISPR‐Cas Systems
Lineage Tracing Using CRISPR‐Cas9
Introduction to Molecular Recording
Molecular Recording Using CRISPR‐Cas9
Molecular Recording Using CRISPR Spacer Acquisition
Outlook
References
Afterword
Index
End User License Agreement
List of Tables
Chapter 2
Table 2.1 The core proteins of CRISPR‐Cas systems
Chapter 6
Table 6.1 Descriptions of type V subtypes
Chapter 8
Table 8.1 CRISPR spacer‐related properties of genomes of lysogens versus no...
Chapter 11
Table 11.1 CRISPR‐Cas enzyme variants for genome editing in mammalian cells...
Chapter 13
Table 13.1 CRISPR screens investigating the interaction of host cells and b...
List of Illustrations
Chapter 1
Figure 1.1 General mechanism of CRISPR‐Cas immunity. The typical CRISP...
Chapter 2
Figure 2.1 The two classes of CRISPR‐Cas systems, their modular organization...
Figure 2.2 Updated classification of class 1 CRISPR‐Cas systems The di...
Figure 2.3 Origin of type III‐E CRISPR‐Cas systems The diagram d...
Figure 2.4 Updated classification of class 2 CRISPR‐Cas systems The sc...
Figure 2.5 Distribution of the 6 types of CRISPR‐Cas systems in the major ar...
Figure 2.6 Origins and evolution of CRISPR‐Cas systems Shown is a hypo...
Chapter 3
Figure 3.1 Cascade structure. (A)
E. coli
type I‐E
cas
operon and CRISPR arra...
Figure 3.2 RNA‐guided DNA interference by Cascade and Cas3. (A) The Eu...
Figure 3.3 CRISPR adaptation. (A) Crystal structure of the prespacer‐loaded ...
Figure 3.4 Genome editing applications by crRNA‐guided Cascade variants....
Chapter 4
Figure 4.1 Biology of type II CRISPR‐Cas systems. (A) Type II CRISPR‐C...
Figure 4.2 Applications based on the Cas9 DNA targeting platform. Catalytica...
Figure 4.3 Cas9 nuclease DNA cleavage mechanism. Apo‐Cas9 protein consists o...
Figure 4.4 Strategies for expanding the Cas9 toolbox. The major experimental...
Chapter 5
Figure 5.1 CrRNA biogenesis by type III CRISPR‐Cas systems. (A) Typica...
Figure 5.2 Transcription‐dependent DNA and RNA degradation during the type I...
Chapter 6
Figure 6.1 Type V Cas proteins. (A) Structures of Cas12a (left) and Cas12b (...
Figure 6.2 Pre‐crRNA processing by type V systems that use only crRNA (A), u...
Figure 6.3 Targets of type V systems. (A) Schematic depicting dsDNA target c...
Chapter 7
Figure 7.1 Cas13 diversity and locus organization. (A) Locus architectures o...
Figure 7.2 Cas13 in defense, including processing, interference, and collate...
Figure 7.3 Comparison of Cas13 structures. Structures of Cas13a (A), Cas13b
Figure 7.4 Applications of Cas13 in cells and for nucleic acid detection. (A...
Chapter 8
Figure 8.1 A “CRISPR trade‐off.” Continuously acquiring CR...
Figure 8.2 CRISPR‐Cas systems as pangenome editors. By acquiring space...
Chapter 9
Figure 9.1 Biology of the bacteriophage life cycle. Phages can adopt two dif...
Figure 9.2 Phage mechanisms to evade restriction‐modification (R‐M) systems....
Figure 9.3 CRISPR‐Cas evasion mechanisms. A schematic summarizing phag...
Figure 9.4 Anti‐CRISPR (Acr) mechanisms. Functionally characterized Ac...
Chapter 10
Figure 10.1 QS regulates CRISPR‐Cas activity. (A) In
Serratia
, SmaIR, ...
Figure 10.2 Global regulators govern CRISPR‐Cas activity by directly control...
Figure 10.3 The role of stress response systems in regulating CRISPR‐Cas act...
Figure 10.4 Archaeal CRISPR‐Cas regulation in
Sulfolobus
. Several reg...
Chapter 11
Figure 11.1 Overview of genome editing technologies. (A) Progression of geno...
Figure 11.2 Genome editing strategies and outcomes exploit endogenous DNA re...
Figure 11.3 Applications of CRISPR‐Cas genome editing in fundamental researc...
Figure 11.4 Therapeutic genome editing with CRISPR‐Cas systems. (A) Ge...
Chapter 12
Figure 12.1 CRISPR‐Cas9 system for gene editing and CRISPR‐dCas9 system for ...
Figure 12.2 CRISPR‐dCas9 based tools for manipulation of gene expression....
Chapter 13
Figure 13.1 Overview of steps in CRISPR screens. (A) A CRISPR screen begins ...
Figure 13.2 Diverse phenotypes queried in CRISPR screens. CRISPR screens hav...
Figure 13.3 Approaches for phenotypic measurement and sgRNA enrichment. (A) ...
Figure 13.4 Extensions of CRISPR screens. (A) Originally, CRISPR genome‐edit...
Figure 13.5 Illuminating bacterial host‐pathogen interactions with CRISPR sc...
Chapter 14
Figure 14.1 CRISPR‐Cas antimicrobials can be used to selectively targe...
Figure 14.2 Mechanisms of potential CRISPR‐Cas antimicrobials. There a...
Figure 14.3 Bacteriophage delivery of CRISPR‐Cas antimicrobials. Bacte...
Figure 14.4 Altering bacteriophage host range. The host specificities of som...
Chapter 15
Figure 15.1 CRISPR provides resistance against bacteriophages. With
Streptoc
...
Figure 15.2 Phage escape of CRISPR‐encoded immunity. With the
Streptoc
...
Figure 15.3 Characterization of CRISPR‐Cas systems. To repurpose and e...
Figure 15.4 Iterative CRISPR immunizations. Host‐phage cocultures can be pas...
Figure 15.5 Iterative buildup of CRISPR immunity in industrial starter cultu...
Figure 15.6 CRISPR‐based bacterial genotyping. The CRISPR locus spacer...
Figure 15.7 CRISPR immunity applications. CRISPR‐Cas systems can be repurpos...
Chapter 16
Figure 16.1 DNA writing by CRISPR‐Cas systems. Diversity of CRISPR‐Cas...
Figure 16.2 Applications of DNA writing. (A) Lineage tracing aims to establi...
Figure 16.3 Examples of molecular recording by CRISPR‐Cas9. (A) Levera...
Figure 16.4 Molecular recording by CRISPR spacer acquisition. (A) Spacer acq...
Guide
Cover Page
Table of Contents
Title Page
Copyright
Contributors
Begin Reading
Index
End User License Agreement
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EDITED BY
Rodolphe BarrangouDepartment of Food, Bioprocessing, and Nutrition Sciences North Carolina State University Raleigh, North Carolina
Erik J. SontheimerRNA Therapeutics InstituteUniversity of Massachusetts Chan Medical SchoolWorcester, Massachusetts
Luciano A. MarraffiniHoward Hughes Medical InstituteChevy Chase, MarylandLaboratory of BacteriologyThe Rockefeller University New York, New York
Copyright © 2022 American Society for Microbiology. All rights reserved.
Copublication by the American Society for Microbiology and John Wiley & Sons, Inc.
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted by law. Advice on how to reuse material from this title is available at http://wiley.com/go/permissions.
The right of Rodolphe Barrangou, Erik J. Sontheimer, and Luciano A. Marraffini to be identified as the editors of this work/the editorial material in this work has been asserted in accordance with law.
Limit of Liability/Disclaimer of Warranty
While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy of completeness of the contents of this book and specifically disclaim any implied warranties or merchantability of fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The publisher is not providing legal, medical, or other professional services. Any reference herein to any specific commercial products, procedures, or services by trade name, trademark, manufacturer, or otherwise does not constitute or imply endorsement, recommendation, or favored status by the American Society for Microbiology (ASM). The views and opinions of the author(s) expressed in this publication do not necessarily state or reflect those of ASM, and they shall not be used to advertise or endorse any product.
Editorial Correspondence:
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Library of Congress Cataloging‐in‐Publication Data applied for
ISBN 9781683670377 (Hardback); ISBN 9781683670384 (Adobe PDF); ISBN 9781683673613 (e‐Pub)
Cover design and illustration by: Owen Design Co.
Interior design by: Susan Brown Schmidler
Illustrations by: Patrick Lane, ScEYEnce Studios
Contributors
Omar O. Abudayyeh
McGovern Institute for Brain Research at MIT
Massachusetts Institute of Technology
Cambridge, MA 02139
Scott Bailey
Department of Biochemistry and Molecular Biology
Department of Biophysics and Biophysical Chemistry
Bloomberg School of Public Health
Johns Hopkins University
Baltimore, MD 21205
Rodolphe Barrangou
CRISPRlab
Department of Food, Bioprocessing and Nutrition Sciences
North Carolina State University
Raleigh, NC 27695
Morgan Quinn Beckett
Department of Biochemistry and Molecular Biology
Department of Biophysics and Biophysical Chemistry
Bloomberg School of Public Health
Johns Hopkins University
Baltimore, MD 21205
David Bikard
Synthetic Biology Group
Department of Microbiology
Institut Pasteur
Paris 75015, France
Joseph Bondy‐Denomy
Department of Microbiology & Immunology
University of California, San Francisco
San Francisco, CA 94143
Aroa Rey Campa
Department of Microbiology and Immunology
Bio‐Protection Research Centre
University of Otago
Dunedin 9054, New Zealand
Crystal Chen
Department of Chemical Engineering
Stanford University
Stanford, CA 94305
Mariia Y. Cherepkova
Department of Biosystems Science and Engineering
ETH Zurich
4058 Basel, Switzerland
Jonathan D. D'Gama
Department of Microbiology
Harvard Medical School
Division of Infectious Diseases
Brigham & Women's Hospital
Boston, MA 02115
Peter C. Fineran
Department of Microbiology and Immunology
Bio‐Protection Research Centre
University of Otago
Dunedin 9054
New Zealand
Jonathan S. Gootenberg
McGovern Institute for Brain Research at MIT
Massachusetts Institute of Technology
Cambridge, MA 02139
Uri Gophna
The Shmunis School of Biomedicine and Cancer Research
Tel Aviv University
Tel Aviv, Israel, 69978
Sutharsan Govindarajan
Department of Microbiology & Immunology
University of California, San Francisco
San Francisco, CA 94143
Patrick D. Hsu
Department of Bioengineering
Innovative Genomics Institute
Center for Computational Biology
University of California, Berkeley
Berkeley, CA 94720
Tautvydas Karvelis
Institute of Biotechnology
Life Sciences Center
Vilnius University
Vilnius, Lithuania LT‐10257
Eugene V. Koonin
National Center for Biotechnology Information
National Library of Medicine
Bethesda, MD 20894
Peter Lotfy
Biological and Biomedical Sciences PhD Program
Harvard Medical School
Division of Gastroenterology, Hepatology, and Nutrition
Boston Children's Hospital
Boston, MA 02115
Broad Institute of MIT and Harvard
Cambridge, MA 02142
Kira S. Makarova
National Center for Biotechnology Information
National Library of Medicine
Bethesda, MD 20894
Luciano A. Marraffini
Laboratory of Bacteriology
Howard Hughes Medical Institute
The Rockefeller University
New York, NY 10065
Jasprina N. Noordermeer
Department of Bioengineering
Stanford University
Stanford, CA 94305
Joseph S. Park
Department of Microbiology
Harvard Medical School
Division of Infectious Diseases
Brigham & Women's Hospital
Boston, MA 02115
Randall J. Platt
Department of Biosystems Science and Engineering
ETH Zurich
4058 Basel
Department of Chemistry
University of Basel
4003, Basel
Switzerland
Lei S. Qi
Department of Chemical and Systems Biology
ChEM‐H Institute
Stanford University
Stanford, CA 94305
Anita Ramachandran
Department of Biochemistry and Molecular Biology
Bloomberg School of Public Health
Johns Hopkins University
Baltimore, MD 21205
Avery Roberts
CRISPRlab
Department of Food, Bioprocessing and Nutrition Sciences
North Carolina State University
Raleigh, NC 27695
Justen Russell
Synthetic Biology Group
Department of Microbiology
Institut Pasteur
Paris 75015, France
Virginijus Siksnys
Institute of Biotechnology
Life Sciences Center
Vilnius University
Vilnius, Lithuania LT‐10257
Leah M. Smith
Department of Microbiology and Immunology
University of Otago
Dunedin 9054, New Zealand
Erik J. Sontheimer
RNA Therapeutics Institute
University of Massachusetts Chan Medical School
Worcester, MA 01605
Tanmay Tanna
Department of Biosystems Science and Engineering
ETH Zurich
4058 Basel, Switzerland
John van der Oost
Wageningen University & Research
Laboratory of Microbiology
6708WE Wageningen
The Netherlands
Matthew K. Waldor
Department of Microbiology
Harvard Medical School
Division of Infectious Diseases
Brigham & Women's Hospital
Howard Hughes Medical Institute
Boston, MA 02115
Yuri I. Wolf
National Center for Biotechnology Information
National Library of Medicine
Bethesda, MD 20894
Jenny Y. Zhang
Department of Microbiology & Immunology
University of California, San Francisco
San Francisco, CA 94143
Preface
The rise and popularity of CRISPR have been associated with technology underpinning genome manipulation. Gene editing represents, by many measures, one of the most important achievements of modern science. The alteration the genetic makeup of an organism, so often at the center of science fiction classics (Ridley Scott's Blade Runner and Steven Spielberg's Jurassic Park, for example), is now a reality. Little did the scientific community imagine that the best tool to do this, Cas9, would originate from the investigation of how bacteria and archaea fend off phages and plasmids that invade them. Microbiologists were probably the least surprised about this development, as bacteria have repeatedly provided a plethora of molecular gadgets with highly useful biotechnological applications. Most notable among these are restriction enzymes. About 40 years before the Cas9 revolution in gene editing, the harnessing of the specific DNA cleavage properties of another bacterial defense system, restriction‐modification, was the central catalyst for another revolution in biomedical sciences: recombinant DNA. Both CRISPR and restriction‐modification are testaments of the importance of basic science, microbiology, and bacterial defenses for the advancement of biotechnology.
While ahead of Cas9 there is an exciting future of genetic engineering and gene therapy, behind Cas9 there is the complex and fascinating world of CRISPR immunity. The first part of this book provides extensive coverage of this unique world. It opens with a brief recollection of the historical events and seminal work that led to the discovery of CRISPR‐Cas systems and their function in adaptive immunity, as well as the features that they have in common (chapter 1) and a description of their tremendous diversity (chapter 2). This is followed by in‐depth explanations of the different molecular mechanisms of immunity behind this diversity (chapters 3 to 7), their impact on the ecology and evolution of prokaryotic populations (chapter 8), the countermeasures developed by phages to evade CRISPR defenses (chapter 9), and the regulation of expression of CRISPR loci (chapter 10). In the second part, the book focuses on the biotechnological applications of CRISPR‐Cas systems and their derived effectors: the widespread gene editing (chapter 11) and gene regulation (chapter 12) techniques, the use of CRISPR screens in microbiology research (chapter 13), the repurposing of CRISPR‐Cas systems to attack the bacterial chromosome as programmable antimicrobials (chapter 14), the exploitation of CRISPR immunity to protect bacterial cultures of industrial value from phage predation (chapter 15), and, last but not least, the development of molecular recording circuits for synthetic biology applications (chapter 16).
Such a comprehensive collection of CRISPR chapters would have been impossible without the generous commitment and invaluable contributions of all the authors. We are grateful for the privileged participation of founders of the CRISPR field (Koonin, Makarova, Siksnys, and van der Oost), technical innovators and high‐profile contributors (Bailey, Gophna, Fineran, Waldor, and Bikard) and rising stars that have advanced and will continue to advance the field and bring it to new heights (Abudayyeh, Bondy‐Denomy, Gootenberg, Hsu, Platt, and Qi). We are personally grateful for their contribution to this book, their collaborative efforts and spirit over the years, and their commitment to the readership.
We also thank the American Society for Microbiology (ASM), not only for publishing this book but also for their constant support of the CRISPR field, especially for featuring numerous microbiological studies of CRISPR‐Cas systems for over 15 years and recognizing early on the potential interest in and impact of the CRISPR literature. Indeed, the ASM has also provided financial support for many of the annual CRISPR meetings organized by researchers of the field. In addition, the Society has organized innumerable symposia and sessions covering the different aspects of CRISPR immunity at its annual general meeting and also at local meetings.
Finally, we thank our families. Every endeavor we take on as scientists, such as attending week‐long meetings afar, writing grants 3 days before the deadline, and embarking in the publication of a book with more than a dozen different authors, sends us away from home and keeps our minds preoccupied with our work. We are indebted to them for their constant understanding, support, and love.
RODOLPHE BARRANGOU
ERIK J. SONTHEIMER
LUCIANO A. MARRAFFINI
Section IDiversity of CRISPR‐Cas Systems
Chapter 1CRISPR‐Cas Systems: Core Features and Common Mechanisms
Rodolphe Barrangou1, Luciano A. Marraffini2,3, and Erik J. Sontheimer4
1CRISPRlab, Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, 27695
2Laboratory of Bacteriology, The Rockefeller University, New York, NY, 10065
3Howard Hughes Medical Institute, The Rockefeller University, New York, NY, 10065
4RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605
Introduction
CRISPR loci were first described as a group of short, repetitive sequences downstream of the iap gene in Escherichia coli K‐12 (1) and were later also identified in many additional bacterial and archaeal genomes (2). As opposed to other DNA repeats that are adjacent to one another, CRISPR repeats were found to be distinctively separated by similarly short sequences of unknown origin. These sequences, known as “spacers,” were shown to match regions of the genomes of bacteriophages and plasmids that invade prokaryotes (3, 4, 5) (known as the target or protospacer), leading to the hypothesis that CRISPR loci could be linked to infection by these elements. In addition, a conserved set of CRISPR‐associated (cas) genes coding for domains harbored by proteins that participate in transactions among nucleic acids (e.g., nucleases, helicases, and integrases) were often found to flank the CRISPR repeat‐spacer arrays (6). Finally, the isolation and sequencing of apparent non‐protein‐coding RNAs from the archaeon Archaeoglobus fulgidus revealed that spacer sequences were transcribed and processed into small RNAs (7). All of these observations, along with in‐depth bioinformatic analyses of prokaryotic genomes, allowed Koonin and Makarova to synthesize and articulate the first model for CRISPR loci as a prokaryotic defense system (8). Specifically, they proposed an RNA interference (RNAi)‐based immunity, in which the small RNAs derived from the spacers would be used by Cas nucleases to find and destroy complementary transcripts, and suggested a mechanism with functional analogies to eukaryotic RNAi.
This initial phase of CRISPR studies, carried out largely in silico, generated intriguing and testable predictions that spawned a period of experimentation to test the bioinformatic hypotheses. In the first such study, Barrangou and colleagues showed that CRISPR‐Cas systems provide spacer‐specific immunity against bacteriophages in Streptococcus thermophilus(9). More importantly, the work also produced the astonishing finding that immunity is acquired: upon infection, new spacers that match a region of the genome of the invading phage are inserted into the CRISPR array. This process, the hallmark of CRISPR immunity, is known in the field as “adaptation,” as it enables the bacterial host to adapt to the environmental stress imposed by the virus. The authors also showed by gene knockout that cas9 (originally known as cas5 or csn1) is necessary for immunity and that cas4 is required for immunization, implicating cas genes in CRISPR‐encoded resistance. In a second study, Andersson and Banfield used metagenomic analysis of CRISPR spacer sequences to assemble viral genomes present in natural acidophilic biofilms (10). The reconstituted genomes revealed extensive recombination events that disrupted phage genome matches with the CRISPR spacers, a result showing the presence of an active arms race between CRISPR systems and phages, further supporting the role of these loci in prokaryotic defense. Later in the same year, the van der Oost group provided experimental evidence that immunity is guided by the small RNAs derived from spacers, known as CRISPR RNAs (crRNAs) (11). These researchers constructed Escherichia coli strains with individual deletions of the cas genes to identify the endoribonuclease responsible for the generation of crRNAs from a large CRISPR precursor transcript (the pre‐crRNA), Cas6 (originally known as CseE). The mutant strain lacking cas6 was unable to produce crRNAs and also failed at providing CRISPR immunity against lambda phage, demonstrating the essentiality of protospacer‐complementary crRNAs for defense. The reliance of CRISPR‐Cas system on this processed RNA guide also established “crRNA biogenesis” as a critical mechanistic step for immunity. Finally, Marraffini and Sontheimer showed that CRISPR loci prevent the transfer of conjugative plasmids in Staphylococcus epidermidis in a spacer‐specific manner (12), demonstrating that CRISPR‐Cas immunity extends beyond phage defense. More importantly, in part through the insertion of a self‐splicing intron within the target sequence, which disrupts the DNA target but not its transcript (splicing reconstitutes the original sequence), they demonstrated that immunity can operate by targeting the DNA, as distinct from the RNA, of the invader. This finding not only contrasted with the original prediction that CRISPR‐Cas systems have an RNAi‐like mechanism but also prompted the proposal that the CRISPR machinery could be repurposed as an RNA‐programmable, sequence‐specific DNA cleavage system, perhaps even beyond prokaryotes (12). Altogether, these early mechanistic studies collectively established CRISPR‐Cas as adaptive, DNA‐encoded (heritable), RNA‐mediated, nucleic acid‐targeting immune systems (Fig. 1.1).
Figure 1.1General mechanism of CRISPR‐Cas immunity. The typical CRISPR locus (top) harbors the CRISPR array of repeats and spacers flanked by a set of cas genes. This set contains cas1 and cas2, which are almost universally conserved and participate in the spacer acquisition process, as well as signature and accessory cas genes that determine the CRISPR type and are usually involved in the later stages of the CRISPR‐Cas immune response, crRNA biogenesis and interference. During spacer acquisition, short pieces of the invader’s DNA are integrated into the CRISPR array, a process that is accompanied by the duplication of a repeat sequence. During crRNA biogenesis, the CRISPR array is transcribed and processed into small RNA guides known as CRISPR RNAs (crRNAs). Finally, crRNAs guide the Cas effector complex to its complementary target nucleic acid. Here, we show the mechanism for types I, II and V CRISPR‐Cas systems, where the crRNA recognizes complementary DNA target sequences that are flanked by a protospacer adjacent motif (PAM). The effector complexes of types III and VI systems use the crRNA to find complementary transcripts of the invader.
After these seminal studies, CRISPR research quickly accelerated. Explorations of the molecular mechanisms of immunity, as well as deep and comprehensive identification and bioinformatic analyses of CRISPR loci deposited in GenBank, led to the recognition of different CRISPR‐Cas types and subtypes (13, 14, 15). Interestingly, each of the experimental studies described above investigated a different CRISPR‐Cas system: the S. thermophilus, E. coli, and S. epidermidis experiments focused on type II‐A, I‐E, and III‐A systems, respectively. However, the findings of these foundational studies are still relevant today because they uncovered fundamental aspects of the CRISPR‐Cas immune response that are common to all types. While the chapters that follow present the details and unique features of each different CRISPR‐Cas type (with the exception of type IV, about which relatively little is known), below we describe some of the unifying features of the CRISPR mechanism of action.
The CRISPR Array
Across all CRISPR‐Cas systems, CRISPR loci are defined by their arrays of short (~40‐bp) DNA repeats separated by similarly short spacer sequences. Common to the great majority of CRISPR loci is the presence of a degenerate repeat at the end of the array, with a handful of nucleotides that differ from the rest of the repeats, typically at the 3′ edge of the very last repeat (6). The repeat sequences, however, vary greatly from one locus to another, without any apparent pattern that correlates with the different CRISPR‐Cas types (though with some variation that correlates with the coevolutionary dynamics between the CRISPR repeat sequences and the Cas proteins that interact with them). Indeed, it is possible to classify CRISPR loci based on their repeat sequences (16), and this can be useful for the study of “orphan” arrays (uncommon loci that lack associated cas genes) or arrays found in unassembled, fragmented genomes (missing their associated cas genes). The number of repeats also varies substantially, with many arrays containing just a few and others a few hundred. To date, the record holder is the bacterium Haliangium ochraceum strain DSM 14365, which harbors 588 repeats (and 587 spacers) (17).
Spacer Acquisition
As mentioned above, one of the hallmarks of CRISPR immunity is that new spacers can be acquired upon infection from the genome of the invader (9). This creates a molecular memory of the pathogen that is used to recognize and neutralize it during subsequent attacks. New spacers are added in a polarized manner adjacent to the first repeat of the locus, also known as the “leader” end due to the presence of an A/T‐rich sequence that immediately precedes this repeat. The insertion of a new spacer results in the duplication of the first repeat. This polarized form of integration creates a temporal, iterative record of infections suffered by the host, and this “recording” has been used to trace the evolutionary history of bacteria and archaea (5). Spacer acquisition thus enables an adaptive, genetically encoded, and heritable immune response that is shared across all CRISPR‐Cas types.
Most CRISPR‐Cas types acquire spacer sequences from DNA molecules. The exceptions are type III systems, which can convert RNA into cDNA before its integration into the CRISPR array (18), effectively capturing RNA molecules as DNA spacers (see chapter 5). In both type I and II systems, free double‐stranded DNA (dsDNA) ends are the preferred substrates for the CRISPR adaptation machinery (19, 20). For both systems, the host's recombination nuclease RecBCD increases the efficiency of spacer acquisition at the free dsDNA ends. As a consequence, hot spots of acquisition are generated between these ends and the first chi site that stops RecBCD degradation (19, 20). Although not yet proven experimentally, it is believed that along its path of degradation from the free dsDNA end to the chi site, random and spontaneous disengagement of RecBCD from its substrate will presumably expose a new free dsDNA end from which spacers can be generated and acquired (21). This mode of action has important consequences for the CRISPR pathway. First, since prokaryotic chromosomes are circular but many of their invaders inject a linear DNA genome with a free dsDNA end, it provides a means to distinguish “self” from “foreign” DNA and avoid autoimmunity. Second, it allows the amplification of the CRISPR‐Cas immune response through a process known as “priming” (22, 23, 24). At least in type I and II systems, the dsDNA break introduced by the Cas nucleases can lead to spacer acquisition from the free dsDNA ends that are generated (24, 25). This is particularly important to counteract the rise of escaper phages containing target mutations that prevent efficient targeting and cleavage by Cas effector nucleases. While these mutations lead to the overall failure of CRISPR immunity, in many cases this still allows a relatively low level of target cleavage, sufficient to create new free dsDNA ends and thus trigger the acquisition of new spacers to neutralize phage escapers.
At the molecular level, spacer acquisition is akin to transposon integration, although the inserted DNA is much smaller than typical transposons. The integration reaction is mediated by cas1 and cas2, the only cas genes universally conserved across all CRISPR‐Cas types (15). Cas1 and Cas2 form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer (26, 27). This complex is loaded with a prespacer DNA (the sequence that will become a spacer after integration into the CRISPR array), usually harboring 3′ overhangs, presumably originating from dsDNA ends. The mechanisms of prespacer loading are not completely elucidated and seem to be different for diverse types (27, 28). In contrast, at least in all studies to date, the integration mechanism is universal, involving the nucleophilic attack of the 3′‐OH group of the prespacer overhang on the minus (bottom) strand of the first repeat, distal to the leader (Fig. 1.1). This leads to the formation of a half‐site intermediate and it is followed by a second nucleophilic attack of the other 3′‐OH overhang on the plus (top) strand at the leader‐repeat border. The fully integrated spacer is flanked by single‐stranded repeat sequences, which are converted into double‐stranded sequences, probably by a DNA polymerase gap‐filling activity, to finalize the complete duplication of the repeat. Finally, there is the mechanism of selection of the first repeat for integration varies across types. In type II systems a leader‐anchoring sequence promotes polarized spacer acquisition (29, 30). In contrast, type I spacer integration typically requires the bacterial integration host factor (31), which binds to the leader sequence and induces a sharp DNA bend, required for the type I Cas1‐Cas2 integrase to catalyze the first integration reaction at the leader‐repeat border.
CRISPR Targets
Spacer sequences can be used to find matching sequences in the large databases of DNA sequences and thus infer the targets of the CRISPR‐Cas immune response. It has been estimated that only ~7% of the known spacers have homologous sequences in the databases (26,364 out of 363,460 unique spacers in reference 32), an observation that probably reflects the lack of sequence information from a majority of the prokaryotic genetic elements in general and from viruses in particular. Most of this small fraction of spacers (~96%) matched the genomes of either bacteriophages or prophages. The rest matched to plasmids (~3%), CRISPR‐cas loci (>1%, especially cas3), and prokaryotic DNA that could not be identified as part of mobile genetic elements (>1%) (32). This distribution highlights the fundamental function of CRISPR‐Cas systems in the defense against prokaryotic viruses (9). Similarly to phages, plasmids are a major category of prokaryotic mobile genetic elements (33), but it is not completely clear why they represent the second most abundant CRISPR target. On one hand, plasmids can carry addiction modules, such as toxin/antitoxin systems, that are costly to the host (34), which would benefit from their elimination via CRISPR immunity. On the other hand, the transfer of plasmids can be also beneficial for the recipient organism, for example, antibiotic‐resistant plasmids in bacterial pathogens (35). At the population level, the limits imposed by CRISPR immunity to both phage infection and plasmid conjugation, two of the major routes of horizontal gene transfer (36), have often led to the hypothesis that CRISPR‐Cas systems represent a barrier to the evolution of bacteria and archaea (12, 37). However, theoretical modeling (38) and computational estimation of the rates of horizontal gene transfer (39) have challenged this idea.
Although matched by a minority of spacers, cas genes represent one of the most interesting targets of CRISPR immunity. The simplest explanation for this observation is that CRISPR‐Cas loci are occasionally encoded by both phage (40, 41) and plasmid (42, 43) genomes, and therefore, cas spacers may appear as part of the general CRISPR response against these elements. But more complex scenarios are also conceivable. For instance, some Escherichia coli strains carry an orphan CRISPR locus consisting of a single spacer targeting cas genes present in the type I‐F systems of other strains (44). Importantly, the sequences of the repeats flanking the spacer are recognized and processed into a crRNA by the type I‐F Cascade complex. Therefore, this orphan CRISPR can repel invasion of mobile elements that carry the type I‐F cas locus (44), which upon entry into the host will produce a targeting complex that will turn against their own genome. This example reveals the presence of evolutionary forces that use CRISPR as a barrier against the horizontal transfer of CRISPR‐cas loci themselves. Finally, the presence of spacers against genes that belong to core (i.e. nonmobile) prokaryotic genomic regions suggests a direct role for CRISPR against the horizontal transfer of such genes, as opposed to preventing their spread indirectly, via the attack of phages and plasmids, the main vehicles for genetic exchange (36). Supporting this role, it has been reported that such spacers can prevent the natural transformation of both Gram‐positive (45) and Gram‐negative (46) bacteria. In addition, new spacers matching archaeal housekeeping genes can be acquired during interspecies mating (47), a process in which cells fuse to form a diploid state containing the full genetic repertoire of both parental cells to facilitate genetic exchange and recombination (48). Although not yet completely clear, these spacers could limit subsequent gene transfer, constraining evolution and thus facilitating archaeal speciation.
RNA‐Guided Nucleases
A second hallmark of the CRISPR‐Cas immune response is the employment of programmable RNA‐guided nucleases. The crRNA generated from the transcription and processing of the CRISPR array is loaded into Cas proteins to form a ribonucleoprotein complex that is able to scan nucleic acids for a sequence complementary to the crRNA guide. After successful base‐pairing, the target sequence is cut, with different mechanisms of cleavage for different types. For example, the crRNA is loaded into the Cas nuclease itself in types II, III, V, and VI, resulting in cleavage near or within the target sequence itself (49, 50, 51, 52, 53, 54). However, in type I systems, the crRNA is part of the Cascade complex, which lacks nuclease activity (11). This complex uses the crRNA to find the target and then recruits the helicase/nuclease Cas3, which goes on to degrade the DNA in the vicinity of the target sequence (55).
A major distinction between crRNA‐guided Cas nucleases is their substrates. Types I, II, and V recognize dsDNA, a process that requires the formation of an R loop, a three‐stranded nucleic acid structure composed of the target DNA:crRNA hybrid and the displaced noncomplementary DNA strand (56, 57, 58, 59, 60, 61). Recently, this form of DNA targeting has been shown (in some type I and type V instances) to specify sites of transposon insertion rather than DNA destruction (62, 63). For either of these functional outcomes, stable R‐loop formation is not possible without the presence of a flanking sequence known as the protospacer adjacent motif (PAM) (53, 64, 65, 66). The PAM requirement is thought to fulfill two roles during DNA targeting. First, it facilitates the recognition of the target sequence within the invader genome. The crRNA‐guided nucleases use a PAM recognition domain (60, 67, 68, 69) to scan DNA for the presence of the motif, and R‐loop formation begins only when the protein‐PAM interaction occurs (70, 71). Annealing of the spacer‐target nucleotides immediately flanking the PAM is critical for the success of R‐loop initial formation, and the presence of mismatches in this region (known as the “seed sequence”) prevents targeting (64, 66). This mechanism limits the number of nonproductive attempts at R‐loop formation, thus enabling rapid interrogation and identification of potential target sites within large DNA molecules. It is also suspected of providing binding energy that can be used to drive initial DNA duplex unwinding. A second function for the PAM is to avoid self‐targeting of the CRISPR locus, which contains the target sequence but is flanked by a CRISPR repeat rather than a PAM. Indeed, given that the crRNA guides are fully complementary to the spacer DNA, in the absence of a PAM requirement, the spacer would be recognized as a target. However, since the spacer sequence is flanked by a repeat in the CRISPR array, which does not contain a PAM, self‐targeting is not possible.
Type III and VI CRISPR‐Cas systems, in contrast, use the crRNA guide to find complementary RNA transcripts produced by the invader (52, 54, 72). In these systems, target recognition triggers diverse nonspecific nuclease activities. During type III immunity, base‐pairing of the crRNA with a target transcript activates the DNase activity of the Cas10 subunit (73) to destroy the template DNA. In addition, the Palm domain of Cas10 is activated and converts ATP into cyclic oligoadenylates that act as second messengers to bind and activate Csm6/Csx1 (74, 75), nonspecific RNases that induce a growth arrest of the host to assist the CRISPR defense (76, 77). Interestingly, this self‐targeting is somewhat reminiscent of abortive infection, preventing infected cells from acting as viral factories. In type VI systems, target recognition by the crRNA guide within Cas13 triggers a nonspecific RNase activity of this nuclease (54, 72), also inducing a dormant state in the host that prevents the propagation of the infecting phage (78). In both types, the target RNA is cleaved shortly after its recognition, resulting in the inactivation of the nonspecific nuclease activities (54, 72, 73), presumably to prevent the irreversible destruction of the host cell. As opposed to DNA targeting systems, the crRNA‐guided endoribonucleases do not appear to require a specific motif, i.e., PAM, to cleave the target RNA (79). It is believed that this may be due to the relatively easier mode of target finding (single‐stranded target, no need for unwinding and R‐loop formation, and high copy number of a target transcript compared to its DNA template) and also to the presence of an intrinsic limitation on self‐targeting. This limitation arises because the CRISPR locus generates crRNAs but not complementary transcripts that would trigger self‐immunity: “self‐target” RNAs would be produced only through transcription of the CRISPR array in the opposite direction to the transcription of the crRNAs. Some level of this transcription, however, could be possible even if accidental, and both type III and VI crRNA‐guided nucleases are inhibited upon recognition of self‐targets. Inhibition relies on the annealing of the repeat sequence downstream of the target (the “antitag”) that is complementary to the portion of the repeat sequence harbored upstream of the spacer sequence in the crRNA (the “tag”) (80, 81). As a consequence of this mechanism, bona fide type III and VI targets are never flanked by antitag sequences.
Evasion of CRISPR Immunity
All bacterial and archaeal immune systems are engaged in an arms race with phages and plasmids and are therefore subject to subversion by these invaders. A great proportion of phages carry anti‐CRISPR inhibitors (Acrs) (82), most of which specifically bind to and prevent the function of the crRNA‐guided nucleases of each different type (covered in chapter 9). In addition to Acrs, mutations present in the phage or plasmid population that affect the recognition of the target by the Cas nucleases allow evasion of the immunity imparted by DNA‐targeting CRISPR‐Cas systems. As explained above, these mutations are present in either the PAM or seed sequences (64, 66). However, depending on the genetic function of the target sequence, some escape mutations are not viable, for example, if they change the structure or activity of an essential phage or plasmid gene. Therefore, some spacers mediate less “escapable,” and thus more robust, immunity than others (83, 84). Importantly, spacer acquisition generates a population of bacteria that harbor numerous different spacer sequences (10, 85, 86, 87, 88). This diversity prevents the rise of escapers (89), as most phages in the population will harbor mutations that only enable escape from the immunity provided by one or two spacer sequences but eventually will infect a host cell harboring a spacer they cannot evade. Interestingly, it has been shown that phage and plasmid recombination systems can induce the accumulation of escape mutations within the targets of type I and II Cas nucleases (90, 91). Presumably, these systems can facilitate the introduction of target mutations through their involvement in the repair of the cleaved DNA. Finally, in contrast to DNA targeting CRISPR‐Cas systems, the growth arrest generated during both type III and VI CRISPR immunity limits the possibilities of genetic escape (78, 79). Although there is a low number of phages within the viral population carrying target mutations that could enable escape from recognition and targeting by Cas nucleases, these mutant phages eventually end up infecting a host in which a previous wild‐type phage triggered a growth arrest. These cells are inhospitable for the propagation of any phage, including those with mutant targets. Accordingly, the diversity of the spacer repertoire seems to be less important to counteract the rise of escaper phages during type III and VI immunity (79), an observation that could have an evolutionary correlation with the very low frequencies of spacer acquisition in these systems.
Conclusions
In summary, all CRISPR‐Cas systems share a common general mechanism centered on the CRISPR‐cas locus. The CRISPR array is used to store genetic information from prokaryotic invaders, mostly phages and plasmids, through the extraction and integration of a short DNA spacer sequence from their genome. This information is passed to the Cas nucleases in the form of a crRNA guide, which is used to find and destroy specific, invasive nucleic acids. CRISPR‐Cas systems are locked in a coevolutionary arms race with their targets, which have evolved a series of countermeasures to evade immunity. There are, however, many different CRISPR‐Cas systems, with different features and mechanisms. The chapters that follow describe in detail the wonderful CRISPR diversity that exists in nature.
References
1.
Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A.
1987. Nucleotide sequence of the
iap
gene, responsible for alkaline phosphatase isozyme conversion in
Escherichia coli
, and identification of the gene product.
J Bacteriol
169
:5429–5433.
2.
Mojica FJ, Díez‐Villaseñor C, Soria E, Juez G.
2000. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria.
Mol Microbiol
36
:244–246.
3.
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD.
2005. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
Microbiology (Reading)
151
:2551–2561.
4.
Mojica FJM, Díez‐Villaseñor C, García‐Martínez J, Soria E.
2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.
J Mol Evol
60
:174–182.
5.
Pourcel C, Salvignol G, Vergnaud G.
2005. CRISPR elements in
Yersinia pestis
acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.
Microbiology (Reading)
151
:653–663.
6.
Jansen R, Embden JD, Gaastra W, Schouls LM.
2002. Identification of genes that are associated with DNA repeats in prokaryotes.
Mol Microbiol
43
:1565–1575.
7.
Tang T‐H, Bachellerie J‐P, Rozhdestvensky T, Bortolin M‐L, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A.
2002. Identification of 86 candidates for small non‐messenger RNAs from the archaeon
Archaeoglobus fulgidus
.
Proc Natl Acad Sci U S A
99
:7536–7541.
8.
Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV.
2006. A putative RNA‐interference‐based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action.
Biol Direct
1
:7
http://dx.doi.org/10.1186/1745-6150-1-7
.
9.
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P.
2007. CRISPR provides acquired resistance against viruses in prokaryotes.
Science
315
:1709–1712.
10.
Andersson AF, Banfield JF.
2008. Virus population dynamics and acquired virus resistance in natural microbial communities.
Science
320
:1047–1050.
11.
Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, Dickman MJ, Makarova KS, Koonin EV, van der Oost J.
2008. Small CRISPR RNAs guide antiviral defense in prokaryotes.
Science
321
:960–964.
12.
Marraffini LA, Sontheimer EJ.
2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.
Science
322
:1843–1845.
13.
Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineau S, Mojica FJM, Wolf YI, Yakunin AF, van der Oost J, Koonin EV.
2011. Evolution and classification of the CRISPR‐Cas systems.
Nat Rev Microbiol
9
:467–477.
14.
Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJM, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV.
2015. An updated evolutionary classification of CRISPR‐Cas systems.
Nat Rev Microbiol
13
:722–736.
15.
Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJM, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV.
2020. Evolutionary classification of CRISPR‐Cas systems: a burst of class 2 and derived variants.
Nat Rev Microbiol
18
:67–83.
16.
Nethery MA, Korvink M, Makarova KS, Wolf YI, Koonin EV, Barrangou R.
2021. CRISPRclassify: repeat‐based classification of CRISPR loci.
CRISPR J
4
:558–574.
17.
Pourcel C, Touchon M, Villeriot N, Vernadet J‐P, Couvin D, Toffano‐Nioche C, Vergnaud G.
2020. CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers.
Nucleic Acids Res
48
:D535–D544.
18.
Silas S, Mohr G, Sidote DJ, Markham LM, Sanchez‐Amat A, Bhaya D, Lambowitz AM, Fire AZ.
2016. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase‐Cas1 fusion protein.
Science
351
:aad4234.
19.
Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R.
2015. CRISPR adaptation biases explain preference for acquisition of foreign DNA.
Nature
520
:505–510.
20.
Modell JW, Jiang W, Marraffini LA.
2017. CRISPR‐Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.
Nature
544
:101–104.
21.
Jakhanwal S, Cress BF, Maguin P, Lobba MJ, Marraffini LA, Doudna JA.
2021. A CRISPR‐Cas9‐integrase complex generates precise DNA fragments for genome integration.
Nucleic Acids Res
49
:3546–3556.
22.
Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E.
2012. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system.
Nat Commun
3
:945.
23.
Swarts DC, Mosterd C, van Passel MWJ, Brouns SJJ.
2012. CRISPR interference directs strand specific spacer acquisition.
PLoS One
7
:e35888
http://dx.doi.org/10.1371/journal.pone.0035888
.
24.
Nussenzweig PM, McGinn J, Marraffini LA.
2019. Cas9 cleavage of viral genomes primes the acquisition of new immunological memories.
Cell Host Microbe
26
:515–526.e6.
25.
Semenova E, Savitskaya E, Musharova O, Strotskaya A, Vorontsova D, Datsenko KA, Logacheva MD, Severinov K.
2016. Highly efficient primed spacer acquisition from targets destroyed by the
Escherichia coli
type I‐E CRISPR‐Cas interfering complex.
Proc Natl Acad Sci U S A
113
:7626–7631.
26.
Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA.
2014. Cas1‐Cas2 complex formation mediates spacer acquisition during CRISPR‐Cas adaptive immunity.
Nat Struct Mol Biol
21
:528–534.
27.
Xiao Y, Ng S, Nam KH, Ke A.
2017. How type II CRISPR‐Cas establish immunity through Cas1‐Cas2‐mediated spacer integration.
Nature
550
:137–141.
28.
Rollie C, Graham S, Rouillon C, White MF.
2018. Prespacer processing and specific integration in a type I‐A CRISPR system.
Nucleic Acids Res
46
:1007–1020.
29.
McGinn J, Marraffini LA.
2016. CRISPR‐Cas systems optimize their immune response by specifying the site of spacer integration.
Mol Cell
64
:616–623.
30.
Wright AV, Doudna JA.
2016. Protecting genome integrity during CRISPR immune adaptation.
Nat Struct Mol Biol
23
:876–883.
31.
Nuñez JK, Bai L, Harrington LB, Hinder TL, Doudna JA.
2016. CRISPR immunological memory requires a host factor for specificity.
Mol Cell
62
:824–833.
32.
Shmakov SA, Sitnik V, Makarova KS, Wolf YI, Severinov KV, Koonin EV.
2017. The CRISPR spacer space is dominated by sequences from species‐specific mobilomes.
mBio
8
:e01397‐17
http://dx.doi.org/10.1128/mBio.01397-17
.
33.
Smillie C, Garcillán‐Barcia MP, Francia MV, Rocha EPC, de la Cruz F.
2010. Mobility of plasmids.
Microbiol Mol Biol Rev
74
:434–452.
34.
Yang QE, Walsh TR.
2017. Toxin‐antitoxin systems and their role in disseminating and maintaining antimicrobial resistance.
FEMS Microbiol Rev
41
:343–353.
35.
Weigel LM, Clewell DB, Gill SR, Clark NC, McDougal LK, Flannagan SE, Kolonay JF, Shetty J, Killgore GE, Tenover FC.
2003. Genetic analysis of a high‐level vancomycin‐resistant isolate of
Staphylococcus aureus
.
Science
302
:1569–1571.
36.
Thomas CM, Nielsen KM.
2005. Mechanisms of, and barriers to, horizontal gene transfer between bacteria.
Nat Rev Microbiol
3
:711–721.
37.
Wheatley RM, MacLean RC.
2021. CRISPR‐Cas systems restrict horizontal gene transfer in
Pseudomonas aeruginosa
.
ISME J
15
:1420–1433.
38.
Westra ER, Levin BR.
2020. It is unclear how important CRISPR‐Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements.
Proc Natl Acad Sci U S A
117
:27777–27785.
39.
Gophna U, Kristensen DM, Wolf YI, Popa O, Drevet C, Koonin EV.
2015. No evidence of inhibition of horizontal gene transfer by CRISPR‐Cas on evolutionary timescales.
ISME J
9
:2021–2027.
40.
Minot S, Sinha R, Chen J, Li H, Keilbaugh SA, Wu GD, Lewis JD, Bushman FD.
2011. The human gut virome: inter‐individual variation and dynamic response to diet.
Genome Res
21
:1616–1625.
41.
Seed KD, Lazinski DW, Calderwood SB, Camilli A.
2013. A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity.
Nature
494
:489–491.
42.
Godde JS, Bickerton A.
2006. The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes.
J Mol Evol
62
:718–729.
43.
Millen AM, Horvath P, Boyaval P, Romero DA.
2012. Mobile CRISPR/Cas‐mediated bacteriophage resistance in
Lactococcus lactis
.
PLoS One
7
:e51663
http://dx.doi.org/10.1371/journal.pone.0051663
.
44.
Almendros C, Guzmán NM, García‐Martínez J, Mojica FJM.
2016. Anti‐
cas
spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR‐Cas I‐F systems.
Nat Microbiol
1
:16081
http://dx.doi.org/10.1038/nmicrobiol.2016.81
.
45.
Bikard D, Hatoum‐Aslan A, Mucida D, Marraffini LA.
2012. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection.
Cell Host Microbe
12
:177–186.
46.
Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ.
2013. Processing‐independent CRISPR RNAs limit natural transformation in
Neisseria meningitidis
.
Mol Cell
50
:488–503.
47.
Turgeman‐Grott I, Joseph S, Marton S, Eizenshtein K, Naor A, Soucy SM, Stachler A‐E, Shalev Y, Zarkor M, Reshef L, Altman‐Price N, Marchfelder A, Gophna U.
2019. Pervasive acquisition of CRISPR memory driven by inter‐species mating of archaea can limit gene transfer and influence speciation.
Nat Microbiol
4
:177–186.
48.
Naor A, Lapierre P, Mevarech M, Papke RT, Gophna U.
2012. Low species barriers in halophilic archaea and the formation of recombinant hybrids.
Curr Biol
22
:1444–1448.
49.
Garneau JE, Dupuis M‐È, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S.
2010. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
Nature
468
:67–71.
50.
Gasiunas G, Barrangou R, Horvath P, Siksnys V.
2012. Cas9‐crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.
Proc Natl Acad Sci U S A
109
:E2579–E2586.
51.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.
2012. A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity.
Science
337
:816–821.
52.
Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP.
2009. RNA‐guided RNA cleavage by a CRISPR RNA‐Cas protein complex.
Cell
139
:945–956.
53.
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F.
2015. Cpf1 is a single RNA‐guided endonuclease of a class 2 CRISPR‐Cas system.
Cell
163
:759–771.
54.
Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DBT, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F.
2016. C2c2 is a single‐component programmable RNA‐guided RNA‐targeting CRISPR effector.
Science
353
:aaf5573
http://dx.doi.org/10.1126/science.aaf5573
.
55.
Westra ER, van Erp PBG, Künne T, Wong SP, Staals RHJ, Seegers CLC, Bollen S, Jore MM, Semenova E, Severinov K, de Vos WM, Dame RT, de Vries R, Brouns SJJ, van der Oost J.
2012. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3.
Mol Cell
46
:595–605.
56.
Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O.
2014. Crystal structure of Cas9 in complex with guide RNA and target DNA.
Cell
156
:935–949.
57.
Zhao H, Sheng G, Wang J, Wang M, Bunkoczi G, Gong W, Wei Z, Wang Y.
2014. Crystal structure of the RNA‐guided immune surveillance Cascade complex in
Escherichia coli
.
Nature
515
:147–150.
58.
