Genome Editing in Drug Discovery E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
GENOME EDITING IN DRUG DISCOVERY A practical guide for researchers and professionals applying genome editing techniques to drug discovery In Genome Editing in Drug Discovery, a team of distinguished biologists delivers a comprehensive exploration of genome editing in the drug discovery process, with coverage of the technology's history, current issues and techniques, and future perspectives and research directions. The book discusses techniques for disease modeling, target identification with CRISPR, safety studies, therapeutic editing, and intellectual property issues. The safety and efficacy of drugs and new target discovery, as well as next-generation therapeutics are also presented. Offering practical suggestions for practitioners and academicians involved in drug discovery, Genome Editing in Drug Discovery is a fulsome treatment of a technology that has become part of nearly every early step in the drug discovery pipeline. Selected contributions also include: * A thorough introduction to the applications of CRISPRi and CRISPRa in drug discovery * Comprehensive explorations of genome-editing applications in stem cell engineering and regenerative medicine * Practical discussions of the safety aspects of genome editing with respect to immunogenicity and the specificity of CRISPR-Cas9 gene editing * In-depth examinations of critical socio-economic and bioethical challenges in the CRISPR-Cas9 patent landscape Perfect for academic researchers and professionals in the biotech and pharmaceutical industries, Genome Editing in Drug Discovery will also earn a place in the libraries of medicinal chemists, biochemists, and molecular biologists.
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Table of Contents
Cover
Title Page
Copyright Page
Preface
List of Abbreviations
List of Contributors
Part 1: Introduction to Drug Discovery and Genome Editing Methods
1 Genome Editing in Drug Discovery
1.1 Introduction
1.2 Genome Engineering
1.3 CRISPR/Cas9
1.4 Applications of CRISPR Cas9 in Drug Discovery
1.5 Concluding Comments
References
2 Historical Overview of Genome Editing from Bacteria to Higher Eukaryotes
2.1 Introduction
2.2 Bacterial DNA Engineering (Recombineering)
2.3 BAC Recombineering
2.4 Metabolic Engineering
2.5 Genetic Engineering in Higher Eukaryotes
2.6 Targeted Endonucleases
2.7 Novel Genome Editing Technologies
2.8 Conclusions
References
3 CRISPR Cas
3.1 Introduction
3.2 CRISPR Biology in a Nutshell
3.3 The Diversity of CRISPR Systems
3.4 CRISPR Systems as the Basis for New Tools in Drug Discovery
3.5 Concluding Remarks
References
4 Commercially Available Reagents and Contract Research Services for CRISPR‐Based Studies
4.1 Introduction
4.2 CRISPR Resources and Reagents for Bespoke Editing and Genetic Screening
4.3
In vivo
CRISPR Screening
4.4 Working with Service Providers for Outsourcing CRISPR Studies
4.5 Considerations on Experimental Design and Controls Required when Outsourcing
4.6 Summary
Acknowledgments
References
5 Computational Tools for Target Design and Analysis
5.1 Introduction
5.2 Various Types of CRISPR Effectors
5.3 Computational Tools for Target Design
5.4 Summary
Funding
References
Part 2: Genome Editing in Disease Modeling
6 Genome Editing in Cellular Disease Models
6.1 Gene Editing and Disease Models in Drug Discovery
6.2 Variety of Cellular Disease Models and Their Improvement with Gene Editing
6.3 Choosing and Designing a Relevant Genetically Engineered Cellular Disease Model
6.4 Technical Considerations of Gene Editing in Cells
6.5 Conclusion
References
7 Utilizing CRISPR/Cas9 Technologies for
in vivo
Disease Modeling and Therapy
7.1 Introduction to CRISPR/Cas9 and
in vivo
Modeling
7.2 CRISPR Editing to Alter Gene Expression
7.3 Choice of Cas9 Species and/or Variant and Ortholog
7.4 Tissue‐Specific CRISPR/Cas9 Gene Editing
7.5 Advantages/Disadvantages of Cas9 Expressing Systems
7.6 Limiting and Detecting Off‐Target Editing
in vivo
7.7 Animal Species
7.8 Delivery Systems of CRISPR/Cas9 Components
in vivo
7.9 Concluding Remarks
References
Part 3: Genome Editing in Target Identification and Validation
8 Pooled CRISPR KO Screens for Target Identification
8.1 Introduction
8.2 Pooled CRISPR‐Cas Screens
8.3 Reagents
8.4 Library Transduction, Maintenance, and Next‐Generation Sequencing
8.5 Screen to Target Selection
8.6
In vivo
CRISPR Screens
8.7 Advanced Functional Genomics Screens
8.8 Selected Applications of Pooled CRISPR‐Cas Screens
8.9 Outlook
References
9 Functional Genomics
9.1 Introduction
9.2 Array Format Technologies
9.3 CRISPR Reagent Delivery Systems
9.4 PreClinical Models in Array Screening
9.5 Phenotypic Screening Readouts
9.6 Bioinformatic Pipeline
References
10 Applications of CRISPRi and CRISPRa in Drug Discovery
10.1 Introduction
10.2 Retooling CRISPR to Repress Gene Expression in Human Cells
10.3 Retooling CRISPR to Activate Gene Expression in Human Cells
10.4 Multiplexed CRISPRi/a Genetic Perturbations
10.5 CRISPRi/a Functional Genomics as a Discovery Modality
10.6 Identifying Gene Targets for the Treatment of Disease Using CRISPRi/a
10.7 Identification of Mechanisms of Response and Resistance to Drugs by CRISPRi and CRISPRa
10.8 CRISPRi/a Genetic Interaction Mapping for Drug Discovery
10.9 Conclusion
References
11 Sequence Diversification Screens with CRISPR‐Cas9‐Guided Base Editors
11.1 Introduction
11.2 CRISPR as a Genetic Screening Method
11.3 Conventional Genetic Loss‐ and Gain‐of‐Function Screens Using CRISPR
11.4 Sequence Diversification Screens Using CRISPR Base Editing
11.5 Applications for Base‐Editor Screening
11.6 Conclusion
Acknowledgements
References
12 Single‐Cell Transcriptomics and Epigenomics for CRISPR‐Mediated Perturbation Studies
12.1 Introduction
12.2 CRISPR‐Based Genetic Screens with Single‐Cell Transcriptomics Readout
12.3 CRISPR‐Based Genetic Screens with Single‐Cell Epigenomics Readout
12.4 Future Perspectives
Acknowledgments
References
Part 4: Therapeutic Genome Editing
13 DNA Repair Pathways in the Context of Therapeutic Genome Editing
13.1 Reprogrammable Nucleases
13.2 DNA Double‐Strand Break Repair Pathways
13.3 Strategies to Improve Knock‐In (KI)
13.4 Genome Editing in Clinical Trials
13.5 Major Safety Considerations in TGE Clinical Trials
13.6 Conclusion
References
14 DNA Base Editing Strategies for Genome Editing
14.1 Introduction
14.2 Base Editor Architectures
14.3 Safety Considerations for Base Editing
14.4 Improving Precision, Efficiency, and Specificity
14.5 Prime Editing and Base Editing
14.6 Choosing the Right Editor
14.7 Therapeutic Uses of Base Editors
14.8 Conclusions
References
15 RNA Base Editing Technologies for Gene Therapy
15.1 Introduction
15.2 RNA Editing Technologies
15.3 Potential Clinical Applications of RNA Editing
15.4 Challenges and Opportunities
15.5 Conclusions
References
16 Genome Editing Applications in Cancer T Cell Therapy
16.1 Introduction
16.2 CAR T Cells
16.3 Gene Editing in T Cells
16.4 Gene Editing in T Cell Therapy
16.5 Conclusion
References
17 Genome‐Editing Applications in Stem Cell Engineering and Regenerative Medicine
17.1 Introduction
17.2 Hematological Disorders
17.3 Neurodegenerative Diseases
17.4 Duchenne Muscular Dystrophy
17.5 Ocular Diseases
17.6 Inborn Errors of Metabolism
17.7 Lysosomal Storage Disorders (LSDs)
17.8 Hereditary Tyrosinemia Type I (HT‐1)
17.9 Ornithine Transcarbamylase Deficiency (OTCD)
17.10 Primary Immune Deficiencies
17.11 Current Challenges in Therapeutic Genome Editing
17.12 Conclusions
Acknowledgements
References
18 Delivery and Formulation Methods for Therapeutic Genome Editing
18.1 Introduction
18.2 Payloads and Modalities
18.3 Delivery Technologies
18.4
In vivo
Delivery Strategies
18.5 Conclusion
References
19 Safety Aspects of Genome Editing
19.1 Introduction
19.2 Immunogenicity
19.3 Delivery‐Dependent Immunogenicity
19.4 Methods to Study Cas9 Immunogenicity
19.5 Mitigation Strategies
19.6 Outlook
References
20 Specificity of CRISPR‐Cas9 Gene Editing
20.1 Introduction
20.2 Detecting Genome‐wide Off‐target Effects
20.3 Reducing Genome‐wide Off‐target Effects
20.4 Other Unwanted Effects: Translocations and Large Deletions
20.5 Clinical Implications and Future Directions
References
Part 5: Intellectual Property Aspects and Future Prospects
21 Key Socio‐Economic and (Bio)Ethical Challenges in the CRISPR‐Cas9 Patent Landscape
21.1 Introduction
21.2 CRISPR‐Cas9 is Attracting Great Interest from Both the Business‐enterprise and Academic Sectors
21.3 The Business‐enterprise Sector is Ever More Interested in Using the New and Less Restrictive Forms of IPR for CRISPR‐Cas9
21.4 The Academic Research Sector has Created an Extremely Competitive CRISPR‐Cas9 Patent Landscape
21.5 Certain Socioeconomic and (Bio)ethical Concerns Connected with the CRISPR‐Cas9 Patent Landscape
21.6 Conclusion
References
22 Emerging Technologies for Genome Editing
22.1 Introduction
22.2 Improving and Expanding the Cas9 Toolbox
22.3 Prime Editing
22.4 Targeted Transposition and Beyond
References
Index
End User License Agreement
List of Tables
Chapter 4
Table 4.1 Major providers of CRISPR reagents.
Table 4.2 Genome editing service providers.
Chapter 5
Table 5.1 Type of CRISPRs and properties.
Table 5.2 Web‐based tools for target design with potential off‐target sites...
Table 5.3 Editing activity prediction tools.
Table 5.4 Editing outcome prediction tools.
Table 5.5 Web tools for analysis.
Chapter 6
Table 6.1 Variety of
in vitro
systems available for cellular disease modeli...
Chapter 7
Table 7.1 Comparison of Cas9 proteins.
Chapter 8
Table 8.1 Some of the available academic and commercial human and mouse poo...
Chapter 9
Table 9.1
CRISPR reagent delivery methods
. General advantages and limitatio...
Chapter 11
Table 11.1 Arrayed versus pooled CRISPR screens.
Chapter 12
Table 12.1 Summary of pooled genetic screens that combine CRISPR perturbati...
Table 12.2 Epigenomic technologies that have been developed for single‐cell...
Chapter 13
Table 13.1 Phase I and II clinical trials with applied designer nucleases a...
Table 13.2 Phase I and II clinical trials with designer nucleases in immuno...
Chapter 15
Table 15.1 A summary of the RNA editing technologies developed so far.
Chapter 16
Table 16.1 A decade of gene‐targeting experiments in primary T cells.
Table 16.2 Knock‐out experiments performed on primary T cells.
Chapter 17
Table 17.1 List of clinical trials for various human diseases using genome‐...
Chapter 18
Table 18.1 Overview of viral vectors used for gene therapy.
Chapter 20
Table 20.1 Summary of in silico off‐target detection tools.
Table 20.2 Summary of genome‐wide methods for detecting CRISPR‐Cas9 off‐tar...
List of Illustrations
Chapter 2
Figure 2.1 Graphical overview of genome engineering technologies (upper pane...
Chapter 3
Figure 3.1
Phases of CRISPR‐mediated immune response
. (
a
) The simplifi...
Figure 3.2
Overview of class 1 and class 2 CRISPR systems
. General compositi...
Figure 3.3
crRNA biogenesis pathways
. (a) depicts a canonical crRNA biogenes...
Figure 3.4
Interference mechanism in class 1 systems
. Panel (
a)
depicts inte...
Figure 3.5 Interference mechanisms in class 2 systems. Representative mechan...
Figure 3.6 Adaptation phase(s) in CRISPR system. Depending on whether invadi...
Figure 3.7 Applications of CRISPR systems beyond genome editing. Fusing cata...
Chapter 4
Figure 4.1 Process workflow for project externalization of genome editing to...
Chapter 6
Figure 6.1 Critical steps for the generation of a relevant cellular disease ...
Chapter 7
Figure 7.1
Overview of different Cas9 variants and depiction of selected del
...
Chapter 8
Figure 8.1 CRISPR screening approaches. To introduce CRISPR edits, a cell po...
Figure 8.2 Synthetic lethality and lineage dependencies discovery using pool...
Figure 8.3 Positive selection vs a negative selection pooled screen.
Figure 8.4 Workflow of pooled CRISPR‐Cas screen analysis pipeline.
Figure 8.5 A multitude of choices to be made for planning cancer‐immunothera...
Chapter 9
Figure 9.1
Genome‐wide arrayed CRISPR screening platform.
Overview of ...
Figure 9.2
Arrayed lentiviral CRISPR screening platform.
Overview of a stand...
Chapter 10
Figure 10.1 dCas9 is a programmable RNA‐guided DNA‐binding platform that can...
Figure 10.2 An approach for pooled CRISPRi/a screens in human cells. Custom ...
Chapter 11
Figure 11.1
General principle of a pooled CRISPR screen.
The example shown i...
Figure 11.2
Top. Workflow of a pooled sequence diversification screen using
...
Figure 11.3
Applications of sequence diversification screens.
(a) The major ...
Chapter 12
Figure 12.1 Schematic representation of scRNA‐seq approaches for evaluating ...
Chapter 13
Figure 13.1 Schematic representation of the major DNA double‐strand break (D...
Figure 13.2 Transcript abundance of DSB sensor proteins across normal tissue...
Figure 13.3 Genome editing strategies utilizing non‐homologous end joining (...
Chapter 14
Figure 14.1 Cytosine and adenine base editing in DNA. (a) Architecture of cy...
Figure 14.2 Decision flow chart for choosing base editing tools.
Chapter 15
Figure 15.1 Brief history of developments in the RNA editing technologies.
Figure 15.2 ADAR proteins structure and function. (a) Chemical reaction cata...
Figure 15.3 Different ADAR‐based RNA editing systems. Top 3 (a–c) use endoge...
Chapter 16
Figure 16.1 A schematic representation of the TCR complex and the three gene...
Chapter 17
Figure 17.1
Timeline of genome‐editing nucleases and genome‐editing research
...
Figure 17.2
Ex vivo genome editing‐based autologous cell replacement therapy
...
Figure 17.3
In vivo genome‐editing approach for various human diseases
Figure 17.4
Genome‐editing approaches to enhance dystrophin levels.
DM...
Chapter 18
Figure 18.1
Three main pillars of delivery methods
. Ex‐vivo manipulation of ...
Figure 18.2
The different components required for therapeutic gene editing
. ...
Figure 18.3
Modified mRNA in a lipid nanoparticle.
Modified mRNA or “mod‐RNA...
Figure 18.4
Internal structure and components of a lipid nanoparticle.
Nucle...
Figure 18.5
Chemical structures of cationic ionizable lipids.
Dlin‐MC3‐DMA o...
Figure 18.6
Chemical structures of polymers for nucleic acid delivery.
Diffe...
Figure 18.7
Different applications require different delivery strategies.
Zi...
Chapter 19
Figure 19.1 Innate and adaptive immune responses to various CRISPR delivery....
Chapter 20
Figure 20.1 Cell‐based off‐target detection methods. (a) IDLV capture. IDLV ...
Figure 20.2 In situ off‐target detection methods. (a) BLESS. Following treat...
Figure 20.3 In vitro off‐target detection methods. (a) Digenome‐/DIG‐seq. Pu...
Figure 20.4 Overview of methods for reduction of genome‐wide off‐targets. (a...
Chapter 22
Figure 22.1 Schematic representation of prime editing.
Guide
Cover Page
Genome Editing in Drug Discovery
Copyright Page
Preface
List of Abbreviations
List of Contributors
Table of Contents
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Genome Editing in Drug Discovery
Edited by
Marcello Maresca
AstraZeneca, BioPharmaceuticals R&D
Mölndal, Sweden
Sumit Deswal
AstraZeneca, BioPharmaceuticals R&D
Mölndal, Sweden
This edition first published 2022.© 2022 John Wiley & Sons, Inc.
All rights reserved. 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 or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.
The right of Marcello Maresca and Sumit Deswal to be identified as the authors of the editorial material in this work has been asserted in accordance with law.
Registered OfficeJohn Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA
Editorial Office111 River Street, Hoboken, NJ 07030, USAFor details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com.
Wiley also publishes its books in a variety of electronic formats and by print‐on‐demand. Some content that appears in standard print versions of this book may not be available in other formats.
Limit of Liability/Disclaimer of WarrantyIn view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
Library of Congress Cataloging‐in‐Publication Data
Names: Maresca, Marcello, editor. | Deswal, Sumit, editor.Title: Genome editing in drug discovery / Marcello Maresca, AstraZeneca, BioPharmaceuticals R&D, Mölndal, Sweden, Sumit Deswal, AstraZeneca, BioPharmaceuticals R&D, Mö¨lndal, Sweden.Description: First edition. | Hoboken, NJ : Wiley, 2022. | Includes index.Identifiers: LCCN 2021025952 (print) | LCCN 2021025953 (ebook) | ISBN 9781119671343 (hardback) | ISBN 9781119671381 (adobe pdf) | ISBN 9781119671398 (epub)Subjects: LCSH: Drug development. | Genetic engineering.Classification: LCC RM301.25 .G46 2022 (print) | LCC RM301.25 (ebook) | DDC 615.1/9–dc23LC record available at https://lccn.loc.gov/2021025952LC ebook record available at https://lccn.loc.gov/2021025953
Cover image: © Yurchanka Siarhei/ShutterstockCover design by Wiley
Preface
The development of CRISPR‐Cas9 for genome engineering has revolutionized the field of genome editing. Many of the cell types and animal models previously very challenging to genetic engineering, can now be engineered with high efficiency and precision using CRISPR‐Cas9‐derived tools. This has led to the development of many novel disease models helping scientists to better understand disease biology as well as providing opportunity to test novel therapeutics. By performing large‐scale functional genomics screens with CRISPR‐Cas9, it is now possible to identify and validate drug targets at a much faster rate and better precision. By assessing gene function comprehensively at large scale and in relevant cell type in early stages, candidate attrition rate is reduced. Genome editing is now part of almost every step in the early part of drug discovery pipeline, from target identification and its validation to mechanistic studies in relevant disease models. In addition, genome editing is used as a promising platform for gene therapy and molecular diagnostics.
We felt a need for a book comprehensively covering these important aspects of genome editing in drug discovery and therapy. Such a book will be of very much interest for those performing genome editing in research institutes or applying genome editing to drug discovery projects at pharmaceutical industries. Such a book will also help students of molecular biology, biotechnology, biochemistry, and pharmaceutical sciences to better understand this very important technology and to exemplify how basic research on a bacterial immune system can have such a big impact in life sciences.
We would like to thank staff at Wiley, especially Jonathan Rose, who first proposed the idea of this book. We would like to thank all the authors who spent their precious time in making this book a reality. Each of the chapter is written by experts on the respective topic working in top pharmaceutical/biotechnology companies or academic institutes.
We would also like to thank colleagues and management at AstraZeneca, Mohammad Bohlooly, Steve Rees, Mike Snowden, and Mene Pangalos for supporting us and allowing us to devote our time for such an academic exercise.
Marcello Maresca and Sumit DeswalGothenburg, SwedenApril 2021
List of Abbreviations
AAV
Adeno-Associated Virus
ABE
Adenosine Base Editors
ADAR
Adenosine deaminases acting on RNA
ASOs
Antisense oligonucleotides
CAR
Chimeric Antigen Receptor
Cas9
CRISPR associated protein 9
CBE
Cytosine base editors
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPRa
CRISPR activation
CRISPRi
CRISPR interference
dCas9
Dead Cas9
DSB
Double strand DNA break
FACS
Fluorescence-activated cell sorting
GEMM
Genetically Engineered Mouse Model
HDR
Homology Directed Repair
iGEM
International Genetically Engineered Machines
IPR
Intellectual Property Rights
LNP
Lipid Nano Particles
NHEJ
Non-homologous end joining
PAM
Protospacer adjacent motifs
pegRNA
Prime editing guide RNA
RNAi
RNA interference
RNP
Ribonucleoprotein
sgRNA
Single guide RNA
siRNA
Small interfering RNA
shRNA
Short hairpin RNA
TALENs
Transcription activator-like effector nucleases
TGE
Therapeutic Genome Editing
ZFNs
Zinc finger nucleases
List of Contributors
Pinar AkcakayaGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Nina AkrapDiscovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Lukas BadertscherGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Sangsu BaeDepartment of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South Korea
Fiona M. BehanFunctional Genomics, R&D GlaxoSmithKline, Stevenage, UK
Nupur BhargavaGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Erik Oude BlenkeAdvanced Drug Delivery, Pharmaceutical Sciences, AstraZeneca R&D, Molndal, Sweden
Alessandro BonettiTranslational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Molndal, Sweden
Rakesh Kantilal ChandodeGene Therapy, Functional and Mechanistic Safety, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Aaron T. ChengFunctional Genomics, R&D GlaxoSmithKline, Upper Providence, PA, USA
Yacine ChérifigenOway, Lyon, France
Matthew CoelhoWellcome Sanger Institute, Cambridge, UK
Sumit DeswalGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Ramy ElgendyTranslational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Molndal, Sweden
Leire Escudero‐IbarzDiscovery Biology, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
Justin EyquemGladstone‐UCSF Institute of Genomic Immunology, San Francisco, CA, USADivision of Hemato‐Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USAandDepartment of Microbiology and Immunology & Parker Institute of Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA
Alexandre FraichardgenOway, Lyon, France
Davide GianniDiscovery Biology, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
Luke A. GilbertDepartments of Urology and Cellular & Molecular Pharmacology, and UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA
Sangam Giri GoswamiGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Antje GrotzGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Pragya GuptaGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Gue‐Ho HwangDepartment of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South Korea
Shashank JaitlyGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Venkata R. KrishnamurthyAdvanced Drug Delivery, Pharmaceutical Sciences, AstraZeneca R&D, Boston, MA, USA
Songyuan LiGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Quinn LuNovel Human Genetics Research Unit, R&D GlaxoSmithKline, Upper Providence, PA, USA
Alexandra MadsenGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Franc MaliFaculty of Social Sciences, University of Ljubljana, Ljubljana, Slovenia
Klio MaratouFunctional Genomics, R&D GlaxoSmithKline, Stevenage, UK
Marcello MarescaGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Roberto NitschGene Therapy, Functional and Mechanistic Safety, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
William A. NybergGladstone‐UCSF Institute of Genomic Immunology, San Francisco, CA, USAandDivision of Hemato‐Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA
Jenna PerssonCRISPR Functional Genomics, Karolinska Institutet and SciLifeLab, Stockholm, Sweden
Martin PeterkaGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Michelle J. PorrittGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Sivaprakash RamalingamGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Steve ReesDiscovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
Amélie RezzagenOway, Lyon, France
Saumyaa SaumyaaTranslational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Molndal, Sweden
Bernhard SchmiererCRISPR Functional Genomics, Karolinska Institutet and SciLifeLab, Stockholm, Sweden
Niklas SelfjordGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Ning SunFunctional Genomics, R&D GlaxoSmithKline, Upper Providence, PA, USA
Saša ŠvikovićGenome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Amir Taheri‐GhahfarokhiQuantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Benjamin J.M. TaylorDiscovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
Priya ThakurGenomics and Molecular Medicine, CSIR‐Institute of Genomics and Integrative Biology, New Delhi, India
Pierre TheureygenOway, Lyon, France
Kader ThiamgenOway, Lyon, France
Sandra WimbergerDiscovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Part 1Introduction to Drug Discovery and Genome Editing Methods
This part provides a historical prospective on developments in the genome editing field. Since CRISPR Cas is currently the method of choice, which in true sense has revolutionized genome editing, a separate chapter on the development of this technology is provided.
1 Genome Editing in Drug Discovery
Steve Rees
Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
1.1 Introduction
The last decade has seen the development of an unprecedented number of new technologies that are being applied to transform our understanding of disease and the subsequent success of drug discovery. The dramatic advances in DNA sequencing technology and the more recent advances in other‐omics technologies including transcriptomics, proteomics, and metabolomics are enabling the understanding of disease at the genetic and cellular level to identify new drug targets, and to identify new disease biomarkers to enable disease segmentation and patient stratification in the clinic. Advances in stem cell technology together with technologies that enable the creation of tissue organoids in the laboratory are allowing the creation of complex models of disease (Lancaster et al. 2013), which together with advances in imaging technology are enabling the drug discovery scientist to better understand the efficacy and safety of potential medicines in preclinical studies. The huge increases in computational power, together with advances in Artificial Intelligence and Machine Learning are allowing drug discovery scientists to extract greater knowledge from these large‐omics datasets, to improve the speed and quality of chemistry design and enable the design of improved clinical studies (Vamathevan et al. 2019). Perhaps, the most impactful of the many new technologies applied in drug discovery in the last decade has been the rapid adoption of CRISPR/Cas9 throughout the drug discovery pipeline to create engineered cellular and animal models of disease to enable the study of the role of new drug targets in disease, alongside the development of CRISPR as a medicine in it’s own right or as a key tool in the creation of cell therapy medicines. Taken together, these and other new technologies have impacted every drug discovery program to enable a better understanding of the role of the drug target in disease and the design of molecules more likely to be safe and efficacious in the clinic. Alongside this, a number of new therapeutic modalities are entering the clinic including antisense onligonucleotide, mRNA, protein, and gene and cell therapies which are leading to a situation where every target becomes amenable to therapeutic manipulation. Taken together, the ability of these technologies to improve our understanding of disease, to create safer medicines and to target those medicines to the patient population most likely to benefit from them is leading to an increase in the success of drug discovery. This has been seen in an increase in the number of new molecular entities approved by the FDA with over 40 new medicines being approved each year between 2011 and 2020 compared with an average of around 20 new medicines approved each year between 2001 and 2010 (Batta et al. 2020). A number of reports also describe an increase in success of drug discovery including a recent publication from colleagues in AstraZeneca. Through the implementation of a new research strategy at AstraZeneca in 2010, success from Candidate Selection to product launch has increased from 4% to 20% while 3 projects are now started in early discovery to deliver a Candidate Drug compared with 5 projects in earlier years (Morgan et al. 2018). While this represents a huge increase in drug discovery productivity, it remains the case that the majority of projects fail with the primary cause of failure in research being due to target validation and in the clinic a lack of efficacy in Phase II clinical studies. In both cases, the root cause of failure is that the hypothesis linking the drug target to disease was incorrect and significant efforts are underway in both academia and industry to continue to increase the level of confidence the drug target at the start of, and throughout a drug discovery program to further increase drug discovery success in all therapeutic areas. Throughout this book, authors will present examples of the application of CRISPR/Cas9 to identify novel drug targets, to understand the role of these targets in disease, and to create cellular and animal model systems to allow the development of new medicines more likely to succeed in the clinic. While we remain within the first decade following the discovery of the ability of CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 systems for the precise editing of mammalian genomes (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013), this technology has become embedded throughout drug discovery research (Fellmann et al. 2017). Throughout this book, authors will discuss the current use of CRISPR/Cas9 to facilitate the development of new medicines, as a medicine in its own right, and as a highly sensitive point of care diagnostic. However, we remain in the infancy of the application of this technology, and its potential to transform our understanding and treatment of disease remains huge.
1.2 Genome Engineering
Genome engineering describes the specific introduction of new genetic elements into target genes to mediate either a disruption of the gene sequence, and consequent loss of gene expression, or a change in the protein sequence of the gene being transcribed. The ability to modify gene sequences at precise locations in the genome arose in the 1990s with the development of meganucleases and Zinc Finger Nuclease (ZFN) technology (Kim et al. 1996; Bibikova et al. 2001). ZFNs are synthetic restriction enzymes created by fusing one or more zinc finger DNA‐binding domains, engineered to target a specific DNA sequence, to a DNA nuclease, typically the restriction enzyme Fok1. ZFNs are able to recognize and cut a specific site in the genome to create a double‐stranded DNA break that can be repaired by non‐homologous end joining (NHEJ) or Homology Directed Repair (HDR) to result in gene inactivation or gene repair following the introduction of a DNA repair template. A boost to the technology came in 2009 with the discovery of transcription activator‐like effector nucleases (TALENs) (Boch et al. 2009; Moscou and Bogdanove 2009). TALENs are synthetic restriction enzymes engineered to cut specific sequences of DNA. These are created by fusing a TAL effector DNA‐binding domain designed to recognize the target DNA sequence with a nuclease to mediate DNA cleavage. TALEN constructs can be introduced into cells to cut the DNA at specific sequences to create a double‐stranded DNA break, that following repair by NHEJ, results in the inactivation of the target gene as a consequence of the introduction of additional DNA sequences as part of the repair process. Furthermore, TALENs can be used to change specific nucleotides within a gene, or to introduce new sequences into the genome following transfection of cells with a TALEN’s construct and a DNA repair template. Following HDR at the DNA cut site, the new sequence, encoded by the DNA repair template, can be introduced into the genome, albeit at a low editing efficiency. ZFNs and TALENs technologies have been used extensively in genome engineering projects in medical research and to modify plant genomes. Furthermore, Zinc Finger technology has been used by Sangamo Therapeutics and others to create genome editing medicines. Treatments for a range of diseases are in development with the most advanced project, a treatment for hemophilia currently in Phase 3 clinical studies. However, the widespread adoption of these technologies has been limited due to the requirement for expertise in protein engineering to create a ZFN or TALEN that precisely targets a specific DNA sequence, the relatively low editing efficiencies observed, and the potential for editing at multiple sites in the genome.
1.3 CRISPR/Cas9
The seminal publications in 2013 describing the ability of CRISPR/Cas9 to edit mammalian genomes have led to an explosion in the ability to make precise genetic changes within mammalian cells and animal models using a variety of CRISPR‐based editing methods (Cong et al. 2013; Mali et al. 2013). Since these publications, there has been a dramatic increase in the efficiency and variety of genome editing methods available with these methods now in widespread use in the pharmaceutical industry and academia for target and drug discovery, alongside the discovery and characterization of a number of new editing enzymes and the development of forms of Cas9 with improved editing activity (Gilbert et al. 2014; Kampmann 2018).
CRISPR/Cas is part of the bacterial immune response system where its natural role is to recognize and destroy non‐host nucleic acid sequences as part of the host immune defense system (Wiedenheft et al. 2012). The commonly used laboratory CRISPR system uses the Cas9 nuclease cloned from Streptococcus pyrogenes (SpCas9) although additional Cas9 enzymes have been cloned and characterized (Acharya et al. 2019). In contrast to TALENs and Zinc Finger technology, CRISPR/Cas9 is simple to use in any laboratory. It does not require protein engineering to create a nuclease able to recognize a specific site within the genome, rather targeting of the Cas9 nuclease to specific sites within the genome is mediated through the design of a specific synthetic guide RNA (sgRNA), complementary in sequence to the region of the genome to be targeted, that positions Cas9 at the target site in the genome to result in the creation of a double‐stranded DNA break. Guide RNA design is very straightforward, indeed a number of public‐domain and commercial design tools have become available for the immediate design of highly specific sgRNAs that can be ordered through the Internet and delivered to the laboratory within days. When the sgRNA is introduced into cells alongside SpCas9, the sgRNA recruits the Cas9 nuclease to a specific site in the genome at which a double‐stranded DNA break is introduced into the genome. This is then repaired using the cells’ endogenous DNA repair machinery, commonly through a process termed NHEJ that introduces a small insertion or deletion into the target gene sequence that results in the expression of a nonfunctional protein. Through changing the sequence of the sgRNA, it is theoretically possible to target Cas9 to any site in the genome. In studies aimed at deletion of the gene of interest, the editing efficiencies seen with CRISPR/Cas9 can exceed 90% and are typically above 50% or more of transfected cells making this a highly efficient tool to study the consequences of a loss of gene function in cells and animal models of disease. Furthermore, due to the simplicity of the system, a single Cas9 enzyme can be introduced into a cell with two or more guide RNA to result in the deletion of large pieces of DNA at a single genetic loci, or the independent deletion of multiple genes in parallel, making CRISPR/Cas9 an highly valuable and flexible tool for the study of gene function in cell and animal models.
The introduction of single nucleotide changes, or the insertion of small sequences of heterologous DNA, into a gene can be mediated through the introduction into a cell of Cas9, a sgRNA and a donor DNA template that contains the new sequence. Following gene repair by HDR, the point mutation or additional gene sequence can be introduced the target gene. In contract to gene deletion which is a highly efficient process, gene editing or repair using HDR is a low‐efficiency process, with typically less than 5% of cells being edited, with much work ongoing to develop methods with improved efficiency. While this is a low‐efficiency process, CRISPR enables precise genetic changes to allow the study of the effect of single nucleotide changes and protein truncation on gene function, and the introduction of affinity or other epitope tags into proteins to allow the study of protein location in cells.
In further applications of the technology, variants of Cas9 have been created in which an enzymatically inactive Cas9, that is no longer able to cut the target DNA, is fused to a transcriptional activator or repressor protein (Gilbert et al. 2014; Kampmann 2018). When recruited to the promoter region of a target gene, these versions of Cas9 are able to mediate an activation or repression of gene expression. Base Editor technology has been developed to address the challenge of creating editing systems with increased efficiency for the introduction of precise genetic changes into genes (Rees and Liu 2018). Base Editors consist of a fusion protein between an enzymatically inactive (one site) Cas9nickase and adenosine or cytosine deaminase. These proteins when introduced into cells alongside a targeting sgRNA mediate the enzymatic modification of a specific nucleotide in the genome. Cytosine base editors mediate the transformation of Cytosine to Thytmidine whereas Adenosine Base Editors mediate the transition from Adenosine to Guanosine. In contrast to the introduction of random indels (insertion/deletions) into cells using Cas9, Base Editors mediate specific editing of the target nucleotide.
A further innovation in gene editing technology arose with the publication of Prime Editing (Anzalone et al. 2019). In this technique, a fusion protein is created between an SpCas9 “nickase,” that rather than creating a double‐stranded break in the genome acts to cut a single strand of the DNA, and a reverse transcriptase. When introduced into cells alongside a “prime editing guide” RNA (pegRNA), the pegRNA targets Cas9 to a precise position in the genome where it creates a single‐stranded break in the DNA strand. In contrast to a sgRNA, the pegRNA encodes both a sequence to target the nuclease to a specific site in the genome and a template RNA sequence to be introduced into the genome. The reverse transcriptase creates a DNA copy of the pegRNA which is then introduced into the genome using the cells’ DNA mismatch repair mechanism, resulting in the insertion of a short piece of DNA. This method has been used to introduce point mutations, new codons, and to insert larger DNA sequences into target genes. This method can again theoretically be used to target any sequence in the genome and in contrast to earlier editing methods can result in highly efficient genome modulation.
The huge interest and range of applications for CRISPR have led to the establishment of a series of new vendor companies able to supply CRISPR reagents, both guide RNAs and editing enzymes, for use by the laboratory scientist. This includes organizations such as Synthego and Horizon Discovery as well as the creation of capability in established reagent supply companies including Merck and Thermo Fisher. As well as supplying CRISPR reagents for use in the scientists’ laboratory, these companies also offer a variety of services including the creation of CRISPR‐edited cell lines and animal models and the completion of Functional Genomic screens. Through the work of these companies, CRISPR technology has become democratized for use by any laboratory competent in basic molecular and cell biology techniques. Some of these commercial reagents are discussed in Chapter 4 of this book.
1.4 Applications of CRISPR Cas9 in Drug Discovery
The ability to precisely edit genomes with CRISPR/Cas9 and other related editing systems has become integral to the identification of new drug targets and the creation of engineered cell and animal models of disease. The impact of CRISPR in drug discovery is discussed at length throughout this book and is briefly introduced here. The field of Functional Genomics has advanced with the generation of whole genome‐wide CRISPR libraries and other reagents that enable the parallel deletion, upregulation, or downregulation of every gene in the genome to ask the question “what is the effect of modulating this gene on the biology of interest?” (Doench 2018). These libraries can be prepared in micro‐titer plate format with each well of the plate containing a cocktail of guide RNAs designed to delete a single gene. When screened against cellular models of disease, it becomes possible to identify specific genes which when modulated affect the biology under study. In addition to use for the identification of new drug targets, Functional Genomic screens are being applied widely to address questions such as the identification of genes that when modulated enable an increase in recombinant protein expression or an increase in the productive uptake of lipid nanoparticles. A further application of Functional Genomic screens in Oncology is the screening of whole genome‐wide CRISPR libraries against multiple cancer cell lines in the presence of known cancer medicines to identify genes that mediate resistance or sensitization to that medicine. Many hundred such screens have been performed at Institutes such as the Broad Institute and the Wellcome Trust Sanger Centre to create public domain databases that describe so‐called sensitivity maps of cancer types to drug action (Behan et al. 2019; Cui et al. 2021). Such studies are enabling the targeting of new medicines to specific tumor types, the identification of likely resistance mechanisms to new medicines, and drug combination opportunities in the clinic.
CRISPR is widely applied to create cellular and animal models of disease, both for the identification of new drug targets and for understanding the efficacy of new drug candidates within a discovery program (Lundin et al. 2020). CRISPR is used to create specific mutations in genes to understand the effect of that mutation on gene function and to introduce molecular tags into genes to track gene expression. The latter approach has been widely adapted to characterize the efficacy of Proteolysis Targeting Chimeras (PROTACs) drugs. PROTACs are a recently discovered class of small‐molecule drugs that rather than inhibiting the function of a drug target, act to degrade the target protein. To understand the efficacy of PROTAC drugs in cellular models of disease, the drug target is typically tagged with a short protein sequence that enables the creation of assays that allow PROTAC‐mediated degradation of the target to be followed in real time in an immortalized cell line or animal model of disease. CRISPR has revolutionized the ability to generate transgenic animal models of disease, both reducing the timelines and number of animals required for the creation of an animal model through the ability to highly efficiently edit the genome of the single cell embryo, while again enabling the creation of complex models of disease not previously possible.
CRISPR is being widely applied in the field of CAR‐T cell therapy both to enable precise insertion of the CAR, but also to identify and delete other T‐cell genes to enable improved efficacy of the cell product (Liu et al. 2017). There is also huge interest in the potential of CRISPR as a medicine in its own right to correct gene mutations in rare and perhaps common diseases and a number of biotechnology companies have been established to bring CRISPR medicines to the clinic, including Editas, CRISPR Therapeutics, Beam Therapeutics, Verve Therapeutics and Intellia. The first clinical studies of medicines to treat β‐thalassemia and Sickle Cell Disease started in 2019 with highly promising results in the first patients, with the first in‐vivo gene editing clinical trials in diseases such as Transthyretin amylodosis in which CRISPR is being used to delete genes in the patient liver, due to start in 2022. Many further projects are in discovery to develop treatments for a range of diseases including α1‐antitrypsin deficiency and Cystic Fibrosis.
Last and perhaps one of the most exciting applications of CRISPR in drug discovery is the potential to create highly sensitive, inexpensive, point‐of‐care diagnostics for the early detection of disease (Chen et al. 2018; Gootenberg et al. 2018; Myhrvold et al. 2018). It is widely accepted, particularly in Oncology, that the probability of patient survival from the disease increases with early disease detection. The creation of diagnostics that detect cancer in stage 1 rather than when symptomatic in stage 3 or 4 will transform our ability to treat and perhaps cure this disease. Two methods have been published, described as SHERLOCK and DETECTR, that offer the potential to create such sensitive DNA diagnostics. While in early development, the potential of these innovations is huge and are being applied more broadly, including for the creation of a diagnostic test for the SARS‐CoV2 virus.
1.5 Concluding Comments
Since the demonstration of the ability of CRISPR/Cas systems to precisely and efficiently edit gene sequences in 2012, CRISPR has become embedded as a routine technique in molecular and cell biology laboratories across the field. New industries have been created to supply CRISPR reagents and CRISPR‐edited cell and animal models to the research scientist, to develop CRISPR medicines and to create CRISPR diagnostics. The applications and impact of CRISPR in drug discovery are discussed at length within this book. Within eight short years, CRISPR has transformed our ability to identify and characterize the role of new drug targets in disease and to create the cell and animal models integral to identify and optimize drug candidates. With the rate of innovation in this field, we can look forward to the development of novel CRISPR systems that increase the efficiency and specificity of gene editing, to the development of transformative CRISPR therapies with the potential to cure severe genetic diseases and to the invention of highly sensitive diagnostics for the early identification and subsequent cure of many common diseases. As we move through the coming decades, the opportunity for CRISPR to improve human health remains enormous.
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2 Historical Overview of Genome Editing from Bacteria to Higher Eukaryotes
Marcello Maresca
Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
2.1 Introduction
Molecular cloning methods have been instrumental for the establishment of the biotechnological industry. The ability to clone any DNA sequence of interest into a DNA vector has been a key technology advancement toward the generation of cellular and animal model of disease and the development of biopharmaceuticals. Traditional molecular cloning methods mostly rely on restriction enzymes‐mediated digestion and ligation of the digested fragments. Classical restriction enzymes recognize a relatively short DNA sequence and as a consequence, they are too unspecific to be used directly for DNA engineering applications in cellula.
Novel improvements in DNA assembly methods combined with the cost reduction and with the increase in accuracy of DNA synthesis processes have led to the possibility of assembling large DNA constructs in vitro. Synthetic genomes will have a key role in future DNA engineering platforms but they will not be discussed in this chapter and in this book, where we will focus on in cellula genome engineering approaches.
In this chapter, I will give a brief description of the advancements in the precise genome editing field starting from observations of single‐stranded oligonucleotides‐mediated repair in yeasts to Recombineering and CRISPR‐Cas9‐dependent editing. These technologies have all greatly expanded the tools and methods that are used to generate disease models and to develop assays for drug discovery (Figure 2.1).
2.2 Bacterial DNA Engineering (Recombineering)
Microbes and microbial‐derived systems have been extensively used for the development of novel DNA engineering tools and for the application of these tools to DNA cloning. Restriction enzymes, recombinase systems such as CRE/Lox, integrases such as ΦC31‐Int, and the Cas9‐CRISPR system have all microbial origin. Recombinases and integrases‐based systems have been extensively used to engineer the mammalian genomes but we will not discuss them in this book that is focusing on scarless genome engineering systems. This chapter will focus on the development of Recombineering for bacterial engineering and its use in genome engineering with particular focus on applications in drug discovery.
The inspiration for the Recombineering (recombination‐mediated DNA engineering) method came from studies in yeast where the possibility to introduce an exogenous DNA cassette in yeast genome was demonstrated in the years 1978–1979. Yeasts DNA recombination methods used homology arms to target a gene without the need of double‐strand breaks (DSBs) generation or the expression of any exogenous protein (Hinnen et al. 1978; Scherer and Davis 1979). It was also demonstrated in yeasts that single‐stranded oligonucleotides with short homology arms to a target sequence are able to promote genomic insertions/deletions/modifications (Bhargava et al. 1999) Unfortunately, this approach does not work efficiently in wild‐type bacteria or in higher eukaryotes. This has limited the utility of single‐strand oligonucleotides‐mediated DNA editing of bacterial and mammalian genomes before the advent of recombineering. Murphy´s and Stewart´s groups showed for the first time that a portable recombination system can be introduced in Escherichia coli to induce recombination in bacteria in a similar fashion to the yeast recombination system but with much higher efficiency (Murphy 1998; Zhang et al. 1998). The portable cassette encodes for an exonuclease (i.e. Redα for the lambda Red system), a DNA annealing protein (i.e. Redβ), and the RecBCD inhibitor (i.e. Redγ). In particular, Stewart´s group showed that this system works with very short homology arms (as short as 30nt) via a peculiar mechanism of single‐strand heteroduplex intermediates at the replication fork (Maresca et al. 2010). This observation paved the way to the use of Recombineering for Precise Genome Editing of Bacterial Genome and for molecular cloning strategies. Recombineering overcomes the limitation of classical restriction/ligation‐based cloning because it does not require the availability of unique restriction sites in the target plasmid and it is specific enough to target the bacterial genome. Therefore, Recombineering has been extensively used for the seamless engineering of large constructs such as bacterial artificial chromosomes (BACs) and for the engineering of bacterial genome.
Figure 2.1 Graphical overview of genome engineering technologies (upper panel) and methods (lower panel) developed during the latest 27 years. A schematic representation of the different technologies or methods is presented, respectively, above or under the timeline arrow.
2.3 BAC Recombineering
The use of recombineering has been particularly important for functional genomics programmes where BAC transgenes or Gene Targeting constructs have been engineered at large scale to generate animal models of disease or to develop libraries of gene tagging. The European Conditional Mouse Mutagenesis (EUCOMM) and Knock‐Out Mouse Programme (KOMP) contributed to the large‐scale generation of conditional gene KO mice that have been extensively used in Drug Discovery. In particular, Skarnes and colleagues developed a large conditional knock‐out mouse library in the framework of the EUCOMM programme by using a high‐throughput gene‐targeting pipeline based on Recombineering (Skarnes et al. 2011). This gene‐targeting pipeline has been greatly facilitated by the development of a high‐throughput strategy of DNA engineering where “recombineered” targeting constructs were used to engineer C57BL/6N mouse embryonic stem cell for the generation of KO mice. This mouse library has been instrumental to understand the function of genes encoded by the mammalian genomes (In vivo) and to validate drug targets.
Another particular relevant example of Recombineering applications in Drug Discovery/Development is the remarkable work by scientists at Regeneron Pharmaceuticals aimed to engineer a humanized mouse model producing human–mouse hybrid antibodies. Their VelociGene platform (Murphy 1998) allowed the generation of multiple knock‐out/knock‐in by an high‐throughput recombineering & gene targeting approach where “recombineered” BACs are inserted in mESC using sequential homologous recombination steps. This led to the replacement of mouse immune genes with human orthologs (Valenzuela et al. 2003).
Finally, a very relevant application of Recombineering is the generation of tagged genes libraries. Gene tagging can potentially overcome the use of high‐affinity antibodies to detect gene expression, but it is limited by the lack of faithful gene activity of tagged protein generated with the use of overexpressed cDNA vectors. BAC transgenes guarantee a quas‐physiological level of gene expression maintaining transgene regulatory element and promoters, although the tagged gene is not integrated in its endogenous locus but in a so‐called third allele. The generation of tagged BAC libraries at scale was greatly simplified by selecting recombineering events in liquid bacterial culture. The potential of this system is exemplified by the generation of genome‐wide BAC libraries for the analysis of protein localization in Daino Rerio and Caenorhabditis elegans (Sarov et al. 2006).
2.4 Metabolic Engineering
The highly diverse chemical structures of Natural Products isolated from microbes or derived semisynthetically from natural intermediates allowed the development of a broad range of different drug activities, including antibiotics and chemotherapeutics.
Genome sequencing data facilitated by the development of Next Generation sequencing platforms indicate that microbial genomes contain an untapped resource of biosynthetic gene cluster that can be exploited to generate novel functions.
Unfortunately, most of these gene clusters are not expressed under normal laboratory growth conditions even when it is possible to grow the natural host in lab environment. In addition, the size of the Biosynthetic gene clusters (reaching up to 200kb) renders the in vitro manipulation of this large clusters difficult (Smanski et al. 2016).
Recombineering or Recombineering‐derived strategies have therefore been an ideal method to characterize and to engineer long gene clusters. In fact, specific gene clusters can be inserted in an heterologous host to facilitate the genetic manipulation of the genes present in these clusters.
An alternative strategy would be the use of endogenous recombineering systems from different hosts to manipulate the particular genes present in the gene cluster (Yin et al. 2015).
Recombineering is rapidly becoming the method of choice to manipulate biosynthetic gene clusters but it is also increasingly used to evolve the bacterial genome as pioneered by the work of Church´s group. Hang HH, Isaacs FJ et al. in their landmark paper of 2019 described the use of recombineering to accelerate bacterial genome evolution by an automated multiplex recombineering strategy that they named MAGE (Wang et al. 2009
