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CRISPR Guide RNA Design Tudor A. Fulga David J. H. F. Knapp Quentin R. V. Ferry

CRISPR Guide RNA Design Tudor A. Fulga David J. H. F. Knapp Quentin R. V. Ferry Editors Methods and Protocols Methods in Molecular Biology 2162 M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the ﬁrst to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed. CRISPR Guide RNA Design Methods and Protocols Edited by Tudor A. Fulga, David J. H. F. Knapp, and Quentin R. V. Ferry Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Editors Tudor A. Fulga Radcliffe Department of Medicine Weatherall Institute of Molecular Medicine University of Oxford Oxford, UK David J. H. F. Knapp Radcliffe Department of Medicine Weatherall Institute of Molecular Medicine University of Oxford Oxford, UK Quentin R. V. Ferry Radcliffe Department of Medicine Weatherall Institute of Molecular Medicine University of Oxford Oxford, UK ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0686-5 ISBN 978-1-0716-0687-2 (eBook) https://doi.org/10.1007/978-1-0716-0687-2 © Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microﬁlms 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 speciﬁc 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, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Preface The ability to target and modify DNA sequences is a critical component of natural regu- latory processes. Such systems can be co-opted to delete, insert, or modify DNA at a speciﬁc site in a user-controlled manner. Hitherto, DNA targeting has been the purview of proteins alone. Early attempts to develop such technologies relied on protein evolution for changing target speciﬁcity. Even second-generation tools such as zinc ﬁnger nucleases and transcrip- tion activator-like effector nucleases (TALENs), for which DNA targeting rules could be deﬁned, required arduous molecular biology to alter target speciﬁcity. The repurposing of the clustered regularly interspaced short palindromic repeats (CRISPR) active immune system from bacteria has alleviated much of these issues. Such CRISPR-based systems have two components: a CRISPR-associated protein (Cas) and a short guide RNA that directs the protein to a genomic region following simple base-paring interactions. This system, exempliﬁed by CRISPR/Cas9, allows targeting of almost any speciﬁc DNA sequence with near single-nucleotide resolution by simply reprogramming the short (~20 base pair) spacer region of the guide RNA. Consequently, DNA modiﬁcations can be produced and validated with unprecedented ease. This has resulted in CRISPR/Cas rapidly becoming a staple technique in most biological laboratories. The massive surge in the scope of this technology has also facilitated a rapid diversiﬁcation of applications and CRISPR systems. Cas9 variants with altered sequence speciﬁcities, target ﬁdelities, and enzymatic activities have been developed, which enabled not only gene editing but also controlling transcriptional activity, modifying epigenetic states, RNA localization, and visualization of genomic loci. The backbone of all of these techniques is one simple molecule: the guide RNA. In this volume, we focus on the CRISPR-associated guide RNA and how it can be designed, modiﬁed, and validated for a broad repertoire of purposes. The chapters fall into several sections. We begin with methods for the computational design of target-speciﬁc guide RNAs. We next discuss chemical modiﬁcations which can be used to improve RNA stability, speciﬁcity, and efﬁciency. From there we cover additional modiﬁcations which can be used to create inducible guide RNAs, append additional functional domains, and express guide RNAs in a conditional manner. Finally, we cover methods for measuring off-target guide RNA activity. Overall, these chapters provide a comprehensive pipeline for guide RNA design, which we hope will become an invaluable resource in applying this powerful technology to basic research and therapeutic applications. We would like to thank all contributors to this volume, as well as the series editor, Prof. John M. Walker, for their work on this valuable volume. Finally, we thank you the readers and wish you the best of luck with your CRISPR adventures. Oxford, UK Tudor A. Fulga David J. H. F. Knapp Quentin R. V. Ferry v Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I IN SILICO DESIGN AND OPTIMIZATION OF GUIDE RNA SEQUENCES 1 Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Florian Heigwer and Michael Boutros 2 Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Gue-Ho Hwang, Jin-Soo Kim, and Sangsu Bae PART II CHEMICALLY-MODIFIED GUIDE RNAS 3 Chemical Modiﬁcation of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells. . . . . . . . . 37 Jenny Shapiro, Adi Tovin, Ortal Iancu, Daniel Allen, and Ayal Hendel 4 Gene Disruption Using Chemically Modiﬁed CRISPR-Cpf1 RNA . . . . . . . . . . . . 49 Moira A. McMahon and Meghdad Rahdar 5 “Split-and-Click” sgRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Lapatrada Taemaitree, Arun Shivalingam, Afaf H. El-Sagheer, and Tom Brown 6 Chimeric DNA–RNA Guide RNA Designs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Shuhan Lu, Ying Zhang, and Hao Yin PART III EXPANDING THE CRISPR TOOLBOX 7 Harnessing tRNA for Processing Ability and Promoter Activity. . . . . . . . . . . . . uploads/Litterature/ crispr-guide-rna-design-methods-and-protocols.pdf