Genome Editing in Drug Discovery

1 Cover

2 Title Page Genome Editing in Drug Discovery Edited by Marcello Maresca AstraZeneca, BioPharmaceuticals R&D Mölndal, Sweden Sumit Deswal AstraZeneca, BioPharmaceuticals R&D Mölndal, Sweden

3 Copyright Page

4 Preface

5 List of Abbreviations

6 List of Contributors

7 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

8 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

9 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

10 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

11 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

12 Index

13 End User License Agreement

1 Chapter 4 Table 4.1 Major providers of CRISPR reagents. Table 4.2 Genome editing service providers.

2 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.