Genome Editing in Animals: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Construction and Evaluation of Zinc Finger Nucleases
1 Introduction
2 Materials
2.1 Plasmids and Bacterial Strains
2.2 Primers
2.3 Enzymes
2.4 Instruments and Kits
2.5 Medium and Buffers
3 Methods
3.1 Construction of the Randomized ZFN Library
3.1.1 Primer Extension
3.1.2 Fragment Purification by Electroelution
3.1.3 BbsI Treatment
3.1.4 Small-Scale Ligation
3.1.5 Large-Scale Ligation
3.1.6 Electroporation
3.2 First B1H Selection
3.2.1 Target Sequence Determination
3.2.2 Construction of pH3U3-Target Vector
3.2.3 Electrocompetent Cell Preparation
3.2.4 Electroporation
3.2.5 Plating to Large Plate
3.2.6 Plasmid Purification
3.3 PCR-Based In Vitro Recombination
3.3.1 Amplification of Individual ZFs
3.3.2 In Vitro Recombination
3.3.3 Ligation
3.4 Second B1H Selection
3.4.1 Construction of pH3U3-Target Vector
3.4.2 Preparation of Electrocompetent US0 (pH3U3) Cells
3.4.3 Electroporation and Plating to Large Plates
3.4.4 Colony PCR
3.5 SSA Assay
3.5.1 Construction of pGL4-SSA
3.5.2 Luc Assay
4 Notes
References
Chapter 2: Updated Overview of TALEN Construction Systems
1 Introduction
2 Summary of TALEN Construction Kits
3 Golden Gate Assembly-Based Systems
3.1 Introduction of Golden Gate Assembly
3.2 Typical Protocol for the Golden Gate Assembly-Based TALEN Construction
3.3 Original Golden Gate Kit from Voytas/Bogdanove Lab
3.4 Accessory Plasmids for the Golden Gate Kit
3.5 Platinum Gate Kit from Yamamoto Lab
3.6 TALE Toolbox from Zhang Lab (Combined Method of Golden Gate Assembly and PCR Amplification)
3.7 Multiple Module Library-Mediated Systems from Ekker Lab and Musunuru/Cowan Lab
4 Other Systems
4.1 TAL Effector Engineering Reagents from Joung Lab (Serial Ligation Method)
4.2 LIC Assembly Kit from Hornung Lab (Ligation-Independent Cloning Method)
4.3 Other Systems Currently Unavailable from Addgene
References
Chapter 3: CRISPR/Cas9
1 Introduction
2 Materials
2.1 sgRNA Design
2.2 Preparation of Cassette DNA
2.3 Preparation of Vector
2.4 Ligation
2.5 Transformation and Confirmation of the Clone
2.6 Validation of gRNA
3 Methods
3.1 sgRNA Design
3.2 Preparation of Cassette DNA
3.3 Preparation of Vector
3.4 Ligation
3.5 Transformation and Confirmation of the Clone
3.6 Validation of gRNA
4 Notes
References
Chapter 4: SNPD-CRISPR: Single Nucleotide Polymorphism-Distinguishable Repression or Enhancement of a Target Gene Expression b...
1 Introduction
2 Materials
2.1 Cas9 Expression Constructs (Fig. 1b-e)
2.2 Generation of sgRNA Expression Construct (Fig. 2a)
2.3 Generation of Reporter Assay Constructs Expressing Firefly Luciferase (Fig. 2b)
2.4 Cell Culture, Transfection, and Luciferase Reporter Assay Reagents
3 Methods
3.1 Generation of Cas9 Expression Constructs
3.1.1 pFLAG-Cas9 and pFLAG-dCas9 (Fig. 1b, c)
3.1.2 pFLAG-dCas9-KRAB (Fig. 1d)
3.1.3 pFLAG-dCas9-VP64 (Fig. 1e)
3.2 Generation of sgRNA Expression Construct
3.3 Generation of Reporter Assay Construct Expressing Target Sequence
3.4 Cell Culture and Transfection
3.5 Dual Luciferase Reporter Assay
4 Notes
References
Chapter 5: Utilizing Large Functional and Population Genomics Resources for CRISPR/Cas Perturbation Experiment Design
1 Introduction
2 Materials
2.1 Natural Variation in Human Population and Other Species
2.1.1 Population Frequencies in Health and Disease
2.1.2 Association with Various Complex Human Traits (GWAS and Fine-Mapping)
2.1.3 Association with Gene Expression (eQTLs)
2.1.4 Natural Variation Across Species (Comparative Genomics)
2.2 Functional Genomics and Perturbation Experiments
2.2.1 Large-Scale Functional Genomics Assay
2.2.2 High-Throughput Reporter Assay
2.2.3 Human Cell Perturbations
2.2.4 Knockout in Mouse and Other Model Organisms
3 Methods
3.1 Choosing Negative Controls
3.2 Gene Prioritization
3.3 Choosing Likely Functional Variants
3.4 Choosing Additional Exploratory Variants
3.5 Constructing a Library
4 Notes
References
Chapter 6: Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA
1 Introduction
2 Materials
2.1 In Vitro Transcription
2.2 Embryo Collection and Culture
2.3 Cytoplasmic Microinjection
2.4 Embryo Transfer
2.5 Assay for Genome Modification
3 Methods
3.1 In Vitro Transcription
3.1.1 In Vitro Transcription of gRNA
3.1.2 In Vitro Transcription of Cas9 RNA
3.2 Embryo Collection and Culture
3.3 Cytoplasmic Microinjection
3.3.1 Preparation of the Microinjection Pipet
3.3.2 Preparation of the Holding Pipet
3.3.3 Cytoplasmic Injection
3.4 Embryo Transfer
3.5 Assay for Genome Modification
4 Notes
References
Chapter 7: Gene Targeting in Mouse Embryonic Stem Cells via CRISPR/Cas9 Ribonucleoprotein (RNP)-Mediated Genome Editing
1 Introduction
2 Materials
2.1 gRNA-Cas9 Ribonucleoprotein (RNP)
2.2 Single-Strand Oligonucleotides or Double-Strand DNA for Gene Knock-In
2.3 Embryonic Stem Cells, Doner Blastocyst, and Feeder Cells
2.4 Electroporation System and Genomic DNA Preparation for PCR Genotyping
2.5 Embryo Microinjection
3 Methods
3.1 Targeting Vector Construction
3.2 Cas9-RNP Complex Preparation
3.3 MEF Preparation
3.4 Gene Targeting of ESC and PCR Genotyping
3.5 Flox Allele Generation
3.6 Preparation of ESC for Blastocyst Injection
3.7 Preparation of Blastocyst and Microinjection of ESC
4 Notes
References
Chapter 8: Generation of Knock-In Mouse by Genome Editing
1 Introduction
2 Materials
2.1 ssODN
3 Methods
3.1 Design of ssODN as Knock-In Targeting DNA
3.1.1 For Nucleotide Substitution
3.1.2 For Target-Insertion of Short-Epitope Sequence
3.2 Preparation of Injection Nucleotides
3.2.1 Dilution of the Purchased ssODN
3.2.2 Preparation of Injection Solution
3.3 Zygote Collection, Microinjection, Embryo Transfer, and Genotyping
4 Notes
References
Chapter 9: Introduction of Genetic Mutations Into Mice by Base Editor and Target-AID
1 Introduction
2 Materials
2.1 PCR to Prepare Templates for In Vitro Transcription
2.2 In Vitro Transcription of BE and Target-AID mRNA
2.3 In Vitro Fertilization
2.4 Microinjection to Mouse Zygotes
2.5 Embryo Transfer Into the Oviduct
2.6 Genotyping of Blastocysts and Mice
2.7 Analysis of Off-Target Mutations
3 Methods
3.1 Preparation of BE3 and Target-AID mRNAs
3.2 Preparation of sgRNAs
3.3 In Vitro Fertilization
3.4 Freezing Fertilized Embryos
3.5 Microinjection of Cytoplasm of Mouse Zygotes to Evaluate Efficiency at Blastocyst Stage (See Note 9)
3.6 Genotyping at Blastocyst Stage
3.7 Embryo Transfer into the Oviduct
3.8 Genotyping of Mice
3.9 Analysis of Off-Target Mutations
4 Notes
References
Chapter 10: Genome Editing in Mouse and Rat by Electroporation
1 Introduction
2 Materials
2.1 Preparation of Mouse and Rat Pronuclear Stage Embryos by In Vitro Fertilization (IVF)
2.2 Preparation of Mouse and Rat Pronuclear Stage Embryos by Natural Mating
2.3 Electroporation of Nucleases Into Intact Pronuclear Stage Embryos (TAKE Method)
2.4 Confirmation and Quantity Measurement of Nucleases in the Embryos after TAKE Method
2.5 Embryo Transfer
2.6 Genotyping of Delivered Pups
3 Methods
3.1 Preparation of Mouse and Rat Pronuclear Stage Embryos by IVF
3.2 Preparation of Mouse and Rat Pronuclear Stage Embryos by Natural Mating
3.3 Electroporation of Nucleases Into Intact Pronuclear Stage Embryos (TAKE Method)
3.4 Confirmation and Quantity Measurement of Nucleases in the Embryos After TAKE Method
3.5 Embryo Transfer
3.6 Genotyping of Delivered Pups
4 Notes
References
Chapter 11: Generation of Floxed Mice by Sequential Electroporation
1 Introduction
2 Materials
2.1 Embryo Collection and Culture
2.2 Genome Editing
2.3 Embryo Transfer
2.4 Assay for Genome Modification
3 Methods
3.1 Embryo Collection and Culture
3.2 gRNA Selection
3.3 Donor loxP ssODNs
3.4 The First Step of Electroporation
3.5 The Second Step of Electroporation
3.6 Embryo Transfer
3.7 Assay for LoxP Insertion
4 Notes
References
Chapter 12: Efficient Detection of Flox Mice Using In Vitro Cre Recombination
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 In Vitro Cre Recombination
2.3 Cloning of Floxed Regions and Sequencing Analysis
3 Methods
3.1 DNA Extraction from Mouse Tail Samples
3.2 In Vitro Cre Recombination
3.2.1 First PCR for Amplification of Floxed Region
3.2.2 In Vitro Cre Recombination of PCR Fragments
3.2.3 Second PCR for Detection of Recombined Fragments
3.3 Cloning of Floxed Regions and Sequencing Analysis
3.3.1 Preparation of Linearized Vector by Inverse PCR
3.3.2 Preparation of Insert
3.3.3 Cloning and Sequencing
4 Notes
References
Chapter 13: VCre/VloxP and SCre/SloxP as Reliable Site-Specific Recombination Systems for Genome Engineering
1 Introduction
2 Materials
2.1 Pertinent Sequence Information
2.2 Locations of VCre Plasmids Containing VCre or SCre
2.3 DNA Sequence of Targeting Vectors
2.4 Useful Plasmids
2.5 Genome Databases
2.6 DNA Sequence Analysis Software
2.7 Online Vector Design and Construction Services
2.8 Gene Synthesis Services
2.9 BAC Genomic DNA Bioresources
3 Methods
3.1 Designing the Targeting Vector
4 Notes
References
Chapter 14: Efficient CRISPR/Cas9-Assisted Knockin of Large DNA Donors by Pronuclear Microinjection During S-Phase in Mouse Zy...
1 Introduction
2 Materials
2.1 Design and Construction of the Donor Vector
2.2 Preparation of Pronuclear-Stage Zygotes
2.3 Pronuclear Injection
3 Methods
3.1 Design and Construction of a Donor Vector
3.2 Preparation of Frozen Pronuclear-Stage Zygotes
3.2.1 Superovulation
3.2.2 Collection of Spermatozoa
3.2.3 Collection of Oocytes
3.2.4 In Vitro Fertilization
3.2.5 Cryopreservation
3.3 Thawing Zygotes
3.4 Pronuclear Injection
3.4.1 Production of Injection Needle
3.4.2 Production of Holding Needle
3.4.3 Preparation of Injection Cocktail
3.4.4 Prepare Injection Chamber
3.4.5 Attaching Holding Needle
3.4.6 Attaching Injection Needle for the Piezo-Assisted Microinjection
3.4.7 Piezo-Assisted Microinjection
3.5 Genotyping
4 Notes
References
Chapter 15: Genome Editing of Murine Liver Hepatocytes by AAV Vector-Mediated Expression of Cas9 In Vivo
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture of Murine Hepatocyte Cell Line and Plasmid Transfection
2.3 DNA Isolation
2.4 Confirmation of Genome Editing Efficiency
2.5 Cell Culture for the Production of AAV Vector
2.6 Transfection for AAV Vector Production
2.7 Purification of AAV Vectors
2.8 AAV Titration by qPCR
2.9 Intravenous Vector Injection into Mice
2.10 Evaluation After Gene Transfer in the Mouse Liver
3 Methods
3.1 Construction of Plasmids with gRNA
3.2 Plasmid Transfection of Murine Hepatocyte Cell Line
3.3 T7 Endonuclease Assay
3.4 Production of AAV Vectors
3.5 AAV Titration by qPCR
3.6 Intravenous Injection and Blood Drawing of the Mouse Through the Jugular Vein
3.7 Confirmation of Genome Editing Efficiency in the Mouse Liver
4 Notes
References
Chapter 16: Nonviral Ex Vivo Genome Editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9
1 Introduction
2 Materials
2.1 Medium
2.2 Magnetic Separation
2.3 Electroporation of Genome Editing Tools
2.4 Evaluation of Genome Editing Efficiency
2.5 Transplantation
2.6 Peripheral Blood Collection
3 Methods
3.1 Preparation of Mouse Lin- Cells
3.2 Preparation of Genome Editing Tools
3.3 Genome Editing of Mouse Lin- Cells Using NEPA21
3.4 Evaluation of DSB Efficiency In Vitro
3.5 Evaluation of Knock-in Efficiency In Vitro
3.6 Evaluation of Genome Editing In Vivo
4 Notes
References
Chapter 17: Genome Editing of Rat
1 Introduction
2 Materials
2.1 Preparation of Pronuclear Stage Embryos by Natural Mating
2.2 Preparation of Pronuclear Stage Embryos by In Vitro Fertilization (IVF)
2.3 Microinjection of Nucleases into Pronuclear Stage Embryos (Microinjection Method)
2.4 Electroporation of mRNAs into Intact Pronuclear Stage Embryos (TAKE Method)
2.5 Embryo Transfer
2.6 Genotyping of Delivered Pups
3 Methods
3.1 Preparation of Pronuclear Stage Embryos by Natural Mating
3.2 Preparation of Pronuclear Stage Embryos by In Vitro Fertilization (IVF)
3.3 Microinjection of Nucleases into Pronuclear Stage Embryos (Microinjection Method)
3.4 Electroporation of mRNAs into Intact Pronuclear Stage Embryos (TAKE Method)
3.5 EmbryoTransfer
3.6 Genotyping of Delivered Pups
4 Notes
References
Chapter 18: A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes
1 Introduction
2 Materials
2.1 Animals
2.2 Superovulations
2.3 IVF
2.4 Electroporation
2.5 Embryo Culture
2.6 Embryo Transfer
2.7 Cervical Dislocation
3 Methods
3.1 Superovulation
3.2 IVF
3.3 Electroporation
3.4 Embryo Culture
3.5 Embryo Transfer
3.6 Cesarean Section
4 Notes
References
Chapter 19: Editing the Genome of the Golden Hamster (Mesocricetus auratus)
1 Introduction
2 Materials
2.1 Preparation of Genome Editing Reagents
2.2 Preparation of Hamsters
2.3 Preparation of i-GONAD Experiments
3 Methods
4 Notes
References
Chapter 20: Gene Targeting in Rabbits: Single-Step Generation of Knockout Rabbits by Microinjection of CRISPR/Cas9 Plasmids
1 Introduction
2 Materials
2.1 Super Ovulation
2.2 Fertilized Oocyte Recovery and Embryo Culture
2.3 Microinjection
2.4 Embryo Transplantation
2.5 Parturition and Lactation
3 Methods
3.1 Search for Target Sequences
3.2 Single-Strand Annealing Assay
3.3 Superovulation
3.4 Collection of Fertilized Oocytes
3.5 Injection of Plasmid DNA
3.6 Embryo Transplantation
3.7 Suture: Completion of Surgery
3.8 Birth of Offspring
4 Notes
References
Chapter 21: Genome Editing of Pig
1 Introduction
2 Materials
2.1 Preparation of Genome Editing Tools
2.1.1 ZFNs
2.1.2 TALENs
2.1.3 CRISPR/Cas9
2.1.4 In Vitro Transcription
2.1.5 Purification of RNA
2.1.6 Nuclease-Based Mutation Detection Assay
2.2 Isolation of Nuclear Donor Cells for SCNT
2.2.1 Preparation for Porcine Fetal Fibroblasts (PFFs) for Gene Knockout
2.2.2 Electroporation
2.2.3 Preparation of Knock-in Donor Template or Vector
2.2.4 Isolation of Genetically Modified Cells (Nuclear Donor)
2.3 Somatic Cell Nuclear Transfer (SCNT)
2.3.1 Preparation of In Vitro Matured (IVM) Oocytes
2.3.2 Nuclear Transfer
2.4 Cytoplasmic Injection
2.4.1 In Vitro Fertilization (IVF) of Porcine Oocytes
2.4.2 Cytoplasmic Injection to Porcine Zygotes
2.5 General Equipment and Plastics
3 Methods
3.1 Preparation of Genome Editing Tools
3.1.1 ZFNs
3.1.2 TALENs
3.1.3 CRISPR/Cas9
3.1.4 In Vitro Transcription
3.1.5 Purification of RNA
3.1.6 Nuclease-Based Mutation Detection Assay (Cleavage Detection Assay)
3.1.7 Construction of Donor Vector for Knock-in
3.2 Isolation of Nuclear Donor Cells for SCNT
3.2.1 Preparation for PFFs for Genetically Modified Cells
3.2.2 Electroporation
3.2.3 Isolation of Gene Knockout Cells (Nuclear Donor)
3.2.4 Isolation of Gene Knock-in Cells (Nuclear Donor)
3.3 Somatic Cell Nuclear Transfer (SCNT)
3.3.1 Preparation of In Vitro Matured (IVM) Oocytes
3.3.2 Nuclear Transfer
3.4 Cytoplasmic Injection
3.4.1 In Vitro Fertilization (IVF) of Porcine Oocytes
3.4.2 Cytoplasmic Injection to Porcine Zygotes
4 Notes
References
Chapter 22: GEEP Method: An Optimized Electroporation-Mediated Gene Editing Approach for Establishment of Knockout Pig Lines
1 Introduction
2 Materials
2.1 Common Materials for Each Step
2.2 Oocyte Collection
2.3 In Vitro Production of Porcine Oocytes/Embryos
2.4 Electroporation-Mediated Gene Editing
3 Methods
3.1 Oocyte Collection and IVM
3.2 IVF
3.3 Electroporation-Mediated Delivery of the CRISPR/Cas9 System into Zygotes and IVC
4 Notes
References
Chapter 23: Genome Editing Mediated by Primordial Germ Cell in Chicken
1 Introduction
2 Materials
2.1 Isolation and In Vitro Culture of PGCs
2.2 PGC Transfection and Selection
2.3 Validation of Genome-Edited PGC
2.4 PGC Transplantation
2.5 Testcross for Germline Transmission
2.6 Validation of Genome-Edited Chicken
3 Methods
3.1 Isolation and In Vitro Culture of PGCs
3.2 PGC Transfection and Selection
3.3 Validation of Genome-Edited PGC
3.4 PGC Transplantation
3.5 Testcross for Germline Transmission
3.6 Validation of Genome-Edited Chickens
3.6.1 Validation of Targeted Gene Insertion and Base Editing
4 Notes
References
Chapter 24: CRISPR-Cas9-Mediated Genome Modifications in Zebrafish
1 Introduction
2 Materials
2.1 Preparation of sgRNA, crRNA, tracrRNA, Cas9 mRNA, and Recombinant Cas9 Protein
2.2 Preparation of Donor Templates
2.3 Microinjection
2.4 Genomic PCR and Sequencing Analysis
3 Methods
3.1 Construction of sgRNA Expression Vectors
3.2 Preparation of sgRNA
3.3 Preparation of Cas9 mRNA
3.4 Preparation (Construction) of dsDNA Donors
3.5 Preparation of lssDNA Donors
3.6 Microinjection
3.7 Preparation of Genomic DNA
3.8 Genomic PCR for the Evaluation of gRNA Efficiency
3.9 Genomic PCR for the Evaluation of Knock-in Events
3.10 Determination of the Integration Site by Sequence Analysis
4 Notes
References
Chapter 25: Genome Editing of Medaka
1 Introduction
2 Materials
2.1 Preparation of Single-Guide RNA (sgRNA)
2.2 Production of Cas9 mRNA
2.3 Detection of Mutations in Injected Embryos and Fin Clips Using Heteroduplex Mobility Assay with an Automated Microchip Ele...
2.4 Microinjection into Fertilized Eggs
2.5 Evaluation of Guide RNA (gRNA) Activity
2.6 Establishment of Gene Knockout (KO) Strains
2.7 Preparation of Donor Plasmids and Single-Guide RNAs for Targeted Integration
2.8 Establishment of Gene Knockin (KI) Strains with Cas9 Protein
2.9 Establishment of Gene Knockin (KI) Strains with Cas9 D10A Nickase Protein
2.10 Establishment of Gene Knockin (KI) Strains with PCR Fragment
3 Methods
3.1 Preparation of Single-Guide (sgRNA)
3.2 Production of Cas9 mRNA
3.3 Detection of Mutations in Injected Embryos and Fin Clips Using Heteroduplex Mobility Assay with an Automated Microchip Ele...
3.4 Microinjection into Fertilized Eggs
3.5 Evaluation of Guide RNA (gRNA) Activity
3.6 Establishment of Gene Knockout (KO) Strains
3.7 Preparation of Donor Plasmids for Targeted Integration
3.8 Establishment of Gene Knockin (KI) Strains with Cas9 Protein
3.9 Establishment of Gene Knockin (KI) Strains with PCR Fragment
4 Notes
References
Chapter 26: CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl
1 Introduction
2 Materials
2.1 sgRNA Synthesis
2.2 Preparation of In Vitro Fertilized Eggs
2.3 Injection of Cas9 RNP
2.4 Genotyping
3 Methods
3.1 Design and In Vitro Synthesis of sgRNA
3.2 Preparation of Cas9 RNP
3.3 Preparation of In Vitro Fertilized Eggs
3.4 Injection of Cas9 RNP
3.5 Genotyping Using the Heteroduplex Mobility Assay
3.6 Genotyping Using Multiplexed Amplicon Sequencing
4 Notes
References
Chapter 27: Genome Editing of Silkworms
1 Introduction
2 Materials
2.1 ZFN-Mediated Knockout
2.2 TALEN-Mediated Knockout
2.3 CRISPR/Cas9-Mediated Knockout
2.4 TAL-PITCh-Mediated Knock-in
3 Methods
3.1 ZFN-Mediated Knockout
3.2 TALEN-Mediated Knockout
3.3 CRISPR/Cas9-Mediated Knockout
3.4 Knock-in Using TAL-PITCh
4 Notes
References
Chapter 28: Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN
1 Introduction
2 Materials
2.1 Animals
2.2 Guide RNAs and Cas9
2.3 Reagents
2.4 Apparatus
2.5 Database and Website
3 Methods
3.1 Dechorionation of Eggs
3.2 Microinjection of CRISPR/Cas9 Components
3.3 Examining Mutation Efficiency
3.4 Tissue-Specific Knockouts
3.5 Construction of TALENs
3.6 Electroporation of Vectors
3.7 Germline Mutagenesis
4 Notes
References
Chapter 29: Genome Editing of C. elegans
1 Introduction
2 Materials
2.1 Plasmids
2.2 C. elegans Preparation
3 Methods
3.1 DNA Construction for Cas9-sgRNA Plasmid
3.2 DNA Construction for Homologous Recombination Template
3.3 Injection
3.4 Screening
4 Notes
References
Index