Оглавление
Группа авторов. Genome Editing in Drug Discovery
Table of Contents
List of Tables
List of Illustrations
Guide
Pages
Genome Editing in Drug Discovery
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: From Bacterial Adaptive Immunity to the Swiss Army Knife of Drug Discovery
3.1 Introduction
3.2 CRISPR Biology in a Nutshell
3.3 The Diversity of CRISPR Systems
3.3.1 crRNA Biogenesis
3.3.2 Interference. 3.3.2.1 Class 1. 3.3.2.1.1 Type I
3.3.2.1.2 Type III
3.3.2.1.3 Type IV
3.3.2.2 Class 2
3.3.2.2.1 Type II
3.3.2.2.2 Type V
3.3.2.2.3 Type VI
3.3.3 Adaptation
3.4 CRISPR Systems as the Basis for New Tools in Drug Discovery
3.4.1 Cas Proteins for Gene Editing
3.4.1.1 Cas9 Variants
3.4.1.2 Cas9 Orthologs
3.4.1.3 The Use of Other Cas Proteins in Genome Editing
3.4.2 Application of Cas Proteins Beyond Genome Editing. 3.4.2.1 dCas9 Fusions
3.4.2.2 RNA Targeting
3.4.2.3 Biochemical Detection
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. 4.2.1 Publicly Available Resources
4.2.2 Cas9 Enzymes
4.2.3 Cas9 Alternatives
4.2.4 Guide RNA Formats and Reagents
4.2.5 CRISPR Libraries: CRISPR KO, CRISPRa, CRISPRi
4.3 In vivo CRISPR Screening. 4.3.1 Pooled In vivo CRISPR Screening in Rodent Models
4.3.2 Considerations for Practice of in vivo CRISPR Screening
4.3.3 Next‐Generation in vivo CRISPR Screening
4.4 Working with Service Providers for Outsourcing CRISPR Studies
4.4.1 Critical Steps for Outsourcing
4.5 Considerations on Experimental Design and Controls Required when Outsourcing
4.5.1 Considerations on Selecting the Appropriate Cellular Host
4.5.2 Considerations on the Gene/Locus to be Edited
4.5.3 Controlling CRISPR Off‐Target Effects (OTEs) and Clonal Variations
4.5.4 Deciding on Specific Quality Control Experiments on Engineered Cells
4.5.4.1 Confirmation of Gene KO at Protein Level
4.5.4.2 Confirmation of Genetic Manipulation at RNA Level
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
5.3.1 Web‐Based Tools for Generating Potential Off‐Target Sites
5.3.1.1 Cas‐OFFinder and Cas‐Designer
5.3.1.2 CHOPCHOP
5.3.1.3 CRISPR‐P 2.0
5.3.1.4 CRISPOR
5.3.1.5 CRISPRdirect
5.3.1.6 E‐CRISP
5.3.1.7 CCTop
5.3.2 Computational Tools for Predicting Editing Activity
5.3.2.1 DeepCpf1
5.3.2.2 DeepSpCas9
5.3.2.3 DeepHF
5.3.2.4 CINDEL
5.3.2.5 SSC
5.3.2.6 Prediction Web Tool by Doench et al. 2016
5.3.2.7 CRISPRscan
5.3.3 Computational Tools for Predicting Edited Outcomes After Gene Editing
5.3.3.1 Microhomology‐Predictor
5.3.3.2 FORECasT
5.3.3.3 inDelphi
5.3.4 Computational Tools for Assessing Editing Outcomes
5.3.4.1 TIDE
5.3.4.2 Cas‐Analyzer
5.3.4.3 CRISPResso2
5.3.4.4 CRISPRAnalyzeR
5.3.4.5 CRISPR‐Sub
5.3.5 Computational Tools for Base Editing and Prime Editing
5.3.5.1 BE‐Designer
5.3.5.2 PE‐Designer
5.3.5.3 PrimeDesign
5.3.5.4 DeepBaseEditor
5.3.5.5 DeepPE
5.3.5.6 BE‐Hive
5.3.5.7 BE‐Analyzer
5.3.5.8 PE‐Analyzer
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
6.1.1 Gene Editing Tools and the CRISPR Revolution
6.1.2 Disease Modeling in Drug Discovery
6.2 Variety of Cellular Disease Models and Their Improvement with Gene Editing
6.2.1 Cell Lines and Primary Cultures
6.2.2 Organoids: a Promising 3D Culture System
6.2.3 Human iPS Cells
6.3 Choosing and Designing a Relevant Genetically Engineered Cellular Disease Model
6.3.1 Criteria for an Educated Choice of Cellular System
6.3.2 Impact of Gene Editing Process on the Long‐Term Viability and Physiology of a Cellular Model
6.3.3 Disease Modeling Through Reproduction of Human Mutations in vitro
6.3.4 Alternative Cellular Models Relevant to Diseases’ Pathophysiology
6.4 Technical Considerations of Gene Editing in Cells
6.4.1 Technical Considerations Related to the in vitro System’s Characteristics
6.4.2 Critical Technical Aspects Linked to CRISPR Gene Editing Tool
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. 7.2.1 Gene Knockout and Knockin to Introduce a Point Mutation or Therapeutic Gene Correction
7.2.2 Activation and Repression of Gene Expression by CRISPRa and CRISPRi
7.2.3 CRISPR Library Screens
7.2.4 Generation of Specific Chromosomal Rearrangements Using CRISPR/Cas9
7.3 Choice of Cas9 Species and/or Variant and Ortholog
7.4 Tissue‐Specific CRISPR/Cas9 Gene Editing. 7.4.1 Varying Delivery Vehicles and Choosing Different Routes of Administration
7.4.2 Tissue‐Specific Promoters, sgRNA Processing Systems, and miRNA Response Elements
7.5 Advantages/Disadvantages of Cas9 Expressing Systems
7.6 Limiting and Detecting Off‐Target Editing in vivo
7.7 Animal Species
7.7.1 Rodents and Small Animals. 7.7.1.1 Genetically Engineered Mouse Models (GEMMs)
7.7.2 Large Animal
7.7.3 Invertebrates
7.8 Delivery Systems of CRISPR/Cas9 Components in vivo
7.8.1 Delivery Vehicles for CRISPR Components. 7.8.1.1 Adenovirus (AdV)
7.8.1.2 Adeno‐Associated Virus (AAV)
7.8.1.3 Ribonucleoprotein (RNP) Complex
7.8.1.4 Lipid Nanoparticle (LNPs)
7.8.2 Delivery Routes for 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
8.3.1 Library Design and Synthesis
8.3.2 Biological Systems for Screening
8.4 Library Transduction, Maintenance, and Next‐Generation Sequencing
8.4.1 Virus Production and Library Infection
8.4.2 Replicates
8.4.3 Positive vs Negative Selection Screen
8.5 Screen to Target Selection. 8.5.1 Statistical Analysis of CRISPR Screens
8.5.2 Gene List to Target Selection
8.6 In vivo CRISPR Screens
8.7 Advanced Functional Genomics Screens
8.8 Selected Applications of Pooled CRISPR‐Cas Screens. 8.8.1 Applications in Oncology. 8.8.1.1 Gene Essentiality Screens
8.8.1.2 Synthetic Lethal Screens
8.8.1.3 Drug Resistance and Synergy Screens
8.8.1.4 Immuno‐Oncology Screens for Cancer Immunotherapy
8.8.2 Applications Beyond Oncology. 8.8.2.1 Phenotypic Screens to Understand Gene Function and Regulation
8.8.2.2 Toxicology Applications
8.8.2.3 Microbiology Applications
8.9 Outlook
References
9 Functional Genomics: Arrayed CRISPR KO Screens
9.1 Introduction
9.2 Array Format Technologies
9.2.1 Genetic Perturbation Libraries
9.2.2 Non‐Genetic Perturbation Libraries
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. 11.3.1 CRISPRko Screens
11.3.2 CRISPRi and CRISPRa Screens
11.4 Sequence Diversification Screens Using CRISPR Base Editing. 11.4.1 A Short History of Base Editors
11.4.2 Base‐Editing Sequence Diversification Screens – CRISPR‐X and CRISPR‐TAM
11.4.3 Recent Developments in Base‐Editing Sequence Diversification
11.4.4 Single‐Cell Transcriptomic Read Out of Sequence Diversification Screens
11.4.5 Combining ABE and CBE Editors
11.4.6 Other CRISPR‐Based Sequence Diversification Methods
11.5 Applications for Base‐Editor Screening. 11.5.1 Studying Drug–Target Interactions (DTI)
11.5.2 Regulatory Elements
11.5.3 Alternative Splicing
11.5.4 Limitations
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
12.2.1 CRISP‐seq
12.2.2 Perturb‐seq
12.2.3 CROP‐seq
12.2.4 Mosaic‐seq
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
13.2.1 Non‐Homologous End Joining (NHEJ)
13.2.2 Microhomology‐Mediated End Joining (MMEJ)
13.2.3 Single‐Strand Annealing (SSA)
13.2.4 Homology‐Directed Repair (HDR)
13.3 Strategies to Improve Knock‐In (KI)
13.3.1 Co‐Enrichment Strategies
13.3.2 Compound‐mediated Inhibition of Non‐Homologous End Joining (NHEJ)
13.3.3 Cell Cycle Manipulation
13.3.4 Modifications of sgRNA and HDR Donors
13.4 Genome Editing in Clinical Trials
13.4.1 Strategies Utilizing Non‐Homologous End Joining (NHEJ)
13.4.2 Strategies Engaging Homology‐Directed Repair (HDR)
13.5 Major Safety Considerations in TGE Clinical Trials
13.5.1 Activation of p53 upon Introduction of DNA Breaks
13.5.2 Off‐Target Activity, Large Deletions, and Complex Rearrangements
13.5.3 Pre‐Existing Immunity to Cas9 Variants
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
14.3.1 Off‐Target DNA Editing
14.3.2 Off‐Target RNA editing
14.4 Improving Precision, Efficiency, and Specificity
14.4.1 Precision
14.4.2 Efficiency
14.4.3 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
15.2.1 Systems Employing Endogenous ADAR Proteins
15.2.1.1 GluR2‐ADAR
15.2.1.2 RESTORE
15.2.1.3 LEAPER
15.2.2 Systems Employing an Engineered Version of ADAR Protein
15.2.2.1 SNAP‐ADAR
15.2.2.2 λN‐ADAR
15.2.2.3 MCP‐ADAR
15.2.2.4 CIRTS
15.2.3 CRISPR‐Based RNA Editing Systems
15.3 Potential Clinical Applications of RNA Editing
15.3.1 Duchenne Muscular Dystrophy
15.3.2 Rett Syndrome
15.3.3 Hearing Loss
15.3.4 Inherited Retinal Degeneration
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
16.2.1 Receptor Structure
16.2.2 Alternative Approaches
16.2.3 Clinical Perspectives
16.2.4 Challenges and Adverse Events
16.2.5 Manufacturing and Costs
16.3 Gene Editing in T Cells
16.3.1 Cassette Design and Gene Targeting Strategies
16.3.2 Delivery of Gene Editing Tools
16.3.3 Nuclease Delivery
16.3.4 Donor Template Delivery
16.3.5 Genetic Screens in T Cells
16.4 Gene Editing in T Cell Therapy. 16.4.1 Limiting GVHD and TCR Mispairing
16.4.2 Limit Immunosuppressive Signaling
16.4.3 Delaying Exhaustion of Intratumoral T Cells
16.4.4 Controlling T Cell Fate with Gene Editing
16.4.5 Broadening CAR Target Antigens in Blood Malignancies
16.4.6 Targeted Integration
16.5 Conclusion
References
17 Genome‐Editing Applications in Stem Cell Engineering and Regenerative Medicine
17.1 Introduction
17.2 Hematological Disorders
17.2.1 β‐hemoglobinopathies: Sickle Cell Disease and β‐thalassemia
17.2.1.1 Genetic Correction of the HBB Gene
17.2.1.2 Increasing γ‐globin Expression
17.2.2 Hemophilia
17.2.3 Human Immunodeficiency Virus
17.2.3.1 Genome Editing Using CCR5 as Target
17.2.3.2 Genome Editing Using CXCR4 as Target
17.3 Neurodegenerative Diseases
17.3.1 Parkinson's Disease
17.3.1.1 iPSCs Cell Therapy. 17.3.1.1.1 Healthy Donor Cell Therapy
17.3.1.1.2 Autologous Genetically Corrected Cell Therapy
17.3.2 Alzheimer's Disease
17.3.2.1 CRISPR/Cas9 Correction in Patient‐Derived iPSCs
17.3.3 Huntington's Disease
17.3.3.1 Haplotype‐Specific Approaches of Inactivating Dominant Mutant Allele
17.3.4 Cystic Fibrosis
17.4 Duchenne Muscular Dystrophy
17.4.1 Approaches to Enhance the Dystrophin Levels Using Genome Editing
17.4.1.1 Exon Deletion
17.4.1.2 Exon Skipping and NHEJ‐Derived Reframing
17.4.1.3 HDR‐Mediated Repairing
17.4.1.4 Base Editing
17.4.1.5 CRISPRa
17.4.2 Using iPSCs Toward DMD Treatment
17.5 Ocular Diseases
17.6 Inborn Errors of Metabolism
17.7 Lysosomal Storage Disorders (LSDs)
17.7.1 Hurler Syndrome Mucopolysaccharidosis Type I (MPS‐I)
17.7.2 Hunter Syndrome or Mucopolysaccharidosis Type II (MPS‐II)
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
18.2.1 Plasmid DNA (pDNA)
18.2.2 RNA (mRNA and sgRNA)
18.2.3 Protein/Ribonucleoprotein Complex
18.3 Delivery Technologies
18.3.1 Viral Vectors
18.3.1.1 Production
18.3.1.2 Smaller Cas9 Variants
18.3.1.3 Split‐Constructs
18.3.1.4 Tissue Tropism
18.3.1.5 Chimeric/Hybrid Capsids and Designer Capsids
18.3.1.6 Site‐Specific Promoters and Inducible Expression
18.3.1.7 Summary
18.3.2 Lipid Nanoparticles (LNPs)
18.3.2.1 Ionizable Cationic Lipid for LNPs
18.3.2.2 PEG‐Lipids
18.3.2.3 Phospholipids and Cholesterol
18.3.2.4 Formulations with Microfluidic Mixing
18.3.2.5 ApoE Adsorption and Tissue Interaction
18.3.3 Non‐LNP‐Based Delivery Systems
18.4 In vivo Delivery Strategies
18.4.1 Clinically Feasible Delivery Strategies
18.5 Conclusion
References
19 Safety Aspects of Genome Editing : Immunogenicity
19.1 Introduction
19.2 Immunogenicity
19.2.1 Pre‐Existing Immunogenicity in Human Adults. 19.2.1.1 CRISPR/Cas9
19.2.1.2 Adeno‐Associated Virus (AAV)
19.2.2 Role of Antigen Presenting Cells (APCs)
19.2.3 Role of T Cells
19.3 Delivery‐Dependent Immunogenicity
19.3.1 Viral Delivery
19.3.1.1 New Antigen Formation Risk
19.3.2 NonViral Delivery
19.4 Methods to Study Cas9 Immunogenicity. 19.4.1 In vitro Assays: ELISA, ELISpot, FluoroSpot, Immunopeptidome
19.4.2 in vivo Strategies
19.5 Mitigation Strategies. 19.5.1 Targeting Immune‐Privileged Organs
19.5.1.1 Evasion from MHC Class I Recognition
19.6 Outlook
References
20 Specificity of CRISPR‐Cas9 Gene Editing
20.1 Introduction
20.2 Detecting Genome‐wide Off‐target Effects
20.2.1 In Silico Off‐target Detection Tools
20.2.2 Cell‐Based Off‐target Detection Methods
20.2.3 In Situ Off‐target Detection Methods
20.2.4 In vitro Off‐target Detection Methods
20.2.5 Detecting Off‐target Effects of CRISPR Tools that Do Not Induce DSBs
20.2.6 Off‐target Detection Method Choice
20.3 Reducing Genome‐wide Off‐target Effects
20.3.1 Improving the Specificity of Cas9 Target Recognition and Cleavage
20.3.1.1 Modification of gRNA Sequence and Chemistry
20.3.1.2 Engineered Cas9 Variants
20.3.2 Competitive Binding and Protein Inhibitors
20.3.3 Limiting Nuclease Exposure
20.4 Other Unwanted Effects: Translocations and Large Deletions
20.5 Clinical Implications and Future Directions
References
Note
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
22.2.1 Engineering the Specificity of Cas9
22.2.2 Increasing the Targeting Scope of Cas9
22.2.3 The Search for Small Cas Proteins
22.2.4 Mitigating the Immunogenicity of Cas9
22.3 Prime Editing
22.4 Targeted Transposition and Beyond
References
Index. a
b
c
d
e
f
g
h
i
k
l
m
n
o
p
q
r
s
t
u
v
w
x
y
z
WILEY END USER LICENSE AGREEMENT
