Methods in Molecular Biology 1311 Magnus Lundgren Emmanuelle Charpentier Peter C. Fineran Editors CRISPR Methods and Protocols M ETHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 CRISPR Methods and Protocols Edited by Magnus Lundgren Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden Emmanuelle Charpentier Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, Sweden; Hannover Medical School, Hannover, Germany Peter C. Fineran Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Editors Magnus Lundgren Emmanuelle Charpentier Department of Cell and Molecular Biology Helmholtz Centre for Infection Research Uppsala University Department of Regulation in Infection Biology Uppsala , Sweden Braunschweig , Germany The Laboratory for Molecular Infection Peter C. Fineran Medicine Sweden (MIMS) Department of Microbiology and Immunology Umeå Centre for Microbial Research (UCMR) University of Otago Department of Molecular Biology Dunedin , New Zealand Umeå University Umeå, Sweden Hannover Medical School Hannover, Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2686-2 ISBN 978-1-4939-2687-9 (eBook) DOI 10.1007/978-1-4939-2687-9 Library of Congress Control Number: 2015937760 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Photo Credit: Magnus Lundgren and Stan Brouns Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Pref ace All cellular organisms have developed strategies to combat parasitic genome invaders. In many bacteria and most archaea, CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats)-Cas ( C RISPR- as sociated proteins) has recently emerged as an adap- tive RNA-mediated defense mechanism that provides cells with acquired resistance to invading foreign genetic elements such as bacteriophages and plasmids. These defense sys- tems are composed of Cas proteins, and one or more CRISPR arrays containing a collec- tion of short repeat-spacer sequences that provide the CRISPR RNAs (crRNAs) that target the foreign genome. Immunity to invading genomes is mediated via a three-stage process: (1) adaptation via acquisition of viral or plasmid DNA-derived spacers into the CRISPR array, (2) expression and maturation of the short crRNAs, and (3) interference with the invading cognate foreign genomes. The repeat-spacer CRISPR array contents and cas genes have undergone an extraordi- narily dynamic rate of evolution leading to a complex diversity in the genetic architectures of the CRISPR-Cas systems. Indeed, currently there are three major types (I–III) consist- ing of more than 11 subtypes. Although the general principles of the three-stage immunity process are conserved, CRISPR-Cas has evolved a wide variety of precise and coordinated mechanisms that are mainly based on the Cas proteins involved. Interestingly, recent struc- tural studies of Cas protein interference complexes in type I and III systems have demon- strated an overall similarity in architecture, despite a large difference in subunit sequence and composition. CRISPR-Cas adaptive immune systems are currently one of the most fl ourishing and fascinating research themes in microbiology and much work has been performed in the last 10 years. Important fi ndings have recently been made towards the understanding of the molecular mechanisms of CRISPR-Cas systems mediating crRNA expression and interfer- ence in many select CRISPR-Cas systems. In the last couple of years, we have also witnessed a rapid growth in our understanding of the process of spacer acquisition, although the mechanistic details are largely unknown. Other details around, for example, CRISPR-Cas regulation and the role of the systems in other processes remain obscure. By their remark- able mechanistic and functional diversity, CRISPR-Cas systems constitute an extraordinary source of proteins for RNA-DNA, DNA-protein, RNA-protein, and protein-protein inter- actions that can be developed for applied use. The applications are diverse and include the ability to repress or activate the transcription of genes of interest. The best known applica- tion to date is genome editing based on the type II CRISPR-Cas9 system. It is not an overstatement that the ability to use a single Cas9 protein with a guide RNA to direct site- specifi c cleavage of target DNA has revolutionized genome engineering in organisms rang- ing from bacteria and yeast to plants and animals. The therapeutic potential of CRISPR-Cas9 has been demonstrated by, e.g., curing genetic defects in adult mice, and there is a large interest in developing CRISPR-Cas9 for gene therapy in humans. These advances have resulted in the CRISPR-Cas9 technology being adopted as a common genetic tool in the scientifi c community in a very short time. v vi Preface The purpose of this Methods in Molecular Biology volume is to provide a list of cutting- edge protocols for the study of CRISPR-Cas defense systems at the genomic, genetic, biochemical, and structural levels. A number of the tools and techniques described in this book have been developed specifi cally for the analysis of CRISPR-Cas; others have been adapted from more standard protocols of DNA, RNA, and protein biology. Here, we wish to offer a list of protocols to any scientist who would like to analyze CRISPR-Cas in their favorite bacterial or archaeal species or use CRISPR-based genetic tools. The large panel of techniques included in this volume should also be of high interest for bacterial or archaeal geneticists who study mobile genetic elements and horizontal gene transfer in general as well as researchers engaged in the analysis of general mechanisms of DNA/protein, RNA/ protein, and protein/protein interactions. This list of methods is complemented by the description of genome editing protocols using the CRISPR-Cas9 technology. Chapter 1 describes and compares methodologies of RNA sequencing to investigate crRNA biogenesis and function. Chapter 2 discusses methods to reconstitute Cas protein complexes in vitro. Chapter 3 details a method to analyze cleavage of precursor crRNA by Cas endonucleases. In Chapter 4 , a methodology to annotate and classify CRISPR-Cas systems is detailed. In Chapter 5 , instructions for use of a web-based bioinformatic tool for the identifi cation of targets of the crRNAs from CRISPR arrays are illustrated. Chapters 6 and 7 describe the use of CRISPRs for quick typing of bacteria. Chapter 8 describes the use of mass spectrometry to analyze crRNA. Chapter 9 describes the ability to use primed adap- tation plasmids to rapidly generate CRISPR arrays with spacers targeting the cloned region of interest. Chapter 10 provides a CRISPR-Cas plasmid transformation assay to quantita- tively assess interference. Two different assays are outlined in Chapter 11 that enables the accurate measurement of Cas complex interactions with DNA in electrophoretic mobility shift assays. Chapter 12 describes the expression and purifi cation of CMR complexes in Sulfolobus . Chapters 13 and 14 detail methods to generate and detect spacer acquisition into CRISPR arrays in laboratory phage-host challenge experiments in bacteria and archaea. A positive selection method to identify genes that are required for CRISPR-Cas activity is outlined in Chapter 15 . Chapters 16 and 17 detail how to assay nuclease activity of Cas proteins while Chapter 18 describes how to perform helicase assays on Cas3. Chapter 19 provides methods for characterizing details of Cas complexes binding to target DNA. In Chapter 20 the bioinformatic workfl ow to use CRISPR spacers to create and analyze viromes from metagenomic data is described. Chapters 21 and 22 detail protocols for tar- geted mutagenesis in zebrafi sh and Drosophila , respectively, using CRISPR-Cas9. Finally, Chapter 23 describes how to use dCas9 for transcriptional repression. In summary, this timely volume provides a broad list