CRISPR-Cas Methods: Volume 2 (Springer Protocols Handbooks)

✍ Scribed by M. Tofazzal Islam (editor), Kutubuddin Ali Molla (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 399
• Category: Library

✦ Synopsis

This second volume provides new and updated methods detailing advancements in CRISPR-Cas technical protocols. Chapters guide readers through protocols on prime editing, base editing, multiplex editing, editing in cell-free extract, in silico analysis of gRNA secondary structure and CRISPR-diagnosis.

Authoritative and cutting-edge, CRISPR-Cas Methods, Volume 2 aims to serves as a laboratory manual providing scientists with a holistic view of CRISPR-Cas methodologies and its practical application for the editing of crop plants, cell lines, nematode and microorganism.

The chapter “CRISPR/Cas9-mediated gene editing in human induced pluripotent stem cells” is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

✦ Table of Contents

Foreword
Preface
Contents
Contributors
Chapter 1: A Complete Methodology for the Instruction of CRISPR-Based Gene Editing Using a Simplified Cell-Free Extract System...
1 Introduction
2 Materials
2.1 In Vitro Reaction
2.1.1 Equipment
2.1.2 Reagents and Buffers
2.2 Cleavage Reaction Gel and NotI RFLP Analysis
2.2.1 Equipment
2.2.2 Reagents
3 Methods
3.1 In Vitro Editing of the lacZ Gene
3.1.1 Cas12a-crRNA Complex (RNP Formation)
3.1.2 Cleavage Reaction of the lacZ Plasmid
3.1.3 Recover and Clean DNA Using a Clean and Concentrator Kit (See Table 1)
3.1.4 Recircularization Reaction
3.1.5 Recover and Clean DNA Using a Clean and Concentrator Kit
3.1.6 Transformation of Edited DNA into Competent E. coli Cells
3.2 Cleavage Gel Reaction to Confirm Cutting of the lacZ Gene
3.3 RFLP Analysis Using NotI Restriction Enzyme
4 Results
5 Discussion
References
Chapter 2: In Silico Analysis of gRNA Secondary Structure to Predict Its Efficacy for Plant Genome Editing
1 Introduction
2 Materials
2.1 Procedure
References
Chapter 3: In Vitro Cas9 Cleavage Assay to Check Guide RNA Efficiency
1 Introduction
2 Materials
2.1 Web Tools/Databases for Sequence Information Acquisition
2.2 Chemicals, Reagents, Buffers, and Kits
2.2.1 Cloning of sgRNA and Construction of CRISPR Vector
2.2.2 In Vitro Transcription
2.2.3 RNA Transcript Purification
2.2.4 Target DNA (Genomic or Plasmid) Isolation
2.2.5 Equipment
2.2.6 In Vitro Cleavage Assay
3 Methods
3.1 sgRNA-Encoding Template Generation
3.1.1 Cloning of Single sgRNA into pRGEB32 Vector
3.2 In Vitro Transcription of a sgRNA
3.2.1 Template Generation for In Vitro Transcription
3.2.2 In Vitro Transcription
3.2.3 Purification of RNA Transcript
3.3 Genomic DNA Isolation from Rice Leaves
3.4 PCR Amplification of Target from gDNA (See Note 7)
3.4.1 Purification of PCR Product
3.5 In Vitro Cleavage of Target DNA with SpCas9 Nuclease
3.5.1 Analysis of Cleavage Product
4 Notes
References
Chapter 4: Efficient Multiplexed CRISPR-Cas12a Genome Editing in Plants
1 Introduction
2 Materials
3 Methods
3.1 Construction of Modular crRNA Expression Cassettes
3.2 Assembly of Multiple crRNAs
3.3 Higher-Order Assembly of Multiple crRNAs
3.4 T-DNA Assembly for Multiplexed Genome Engineering
4 Notes
References
Chapter 5: CRISPR-dCas9-Based Targeted Manipulation of DNA Methylation in Plants
Abbreviations
1 Introduction
2 Materials
2.1 Plasmids
2.2 Organisms
2.3 Reagents
2.4 Instruments
2.5 Horticultural Supplies
3 Methods
3.1 Description of the SunTag System Modules
3.1.1 Module 1: dCas9-Fused to Epitope Tails
3.1.2 Module 2: GCN4 Single-Chain Variable Fragment Antibody Fused to NtDRMcd or hTET1cd
3.1.3 Module 3: gRNA Expression Cassette
3.2 Workflow
3.2.1 Step 1: Obtaining the SunTag System from Addgene
3.2.2 Step 2: Generating a Custom SunTag Vector
3.2.3 Step 3: Generating and Screening Transgenic Arabidopsis T1 Plants
3.2.4 Step 4: Genotyping
3.2.5 Step 5: Detection of DNA Methylation
3.2.6 Step 6: Analyzing the Heritability of DNA Methylation Changes Induced by the SunTag Systems
4 Conclusion
5 Notes
References
Chapter 6: Rapid Assembly of Multiplex Natural CRISPR Arrays
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 7: Assembly and Assessment of Prime Editing Systems for Precise Genome Editing in Plants
1 Introduction
2 Materials
3 Methods
3.1 T-DNA Vector Construction for Prime Editing with the PE2 System
3.2 Rice Protoplast Transformation to Test Prime Editing Efficiency
4 Notes
References
Chapter 8: Designing, Performing, and Analyzing CRISPR-Cas9-Mediated Genome Editing Experiments in Leguminous Plants
Abbreviations
1 Introduction
2 Choice of CRISPR-Cas9 System
3 Selection of Cas Protein
4 Design of gRNA
5 Choice of Promoters to Express Cas9 and sgRNA
5.1 Plant Transformation
5.2 Materials
5.3 Methods
5.3.1 Preparation of Explants
5.3.2 Half Seed Transformation Protocol
6 Screening of mutants
6.1 qPCR
6.2 High-Resolution Melting Analysis (HRMA)
6.3 Surveyor Nuclease and T7 Endonuclease I
6.4 PCR/Restriction Enzyme Digestion Assay
6.5 PCR/Sequencing
6.6 Whole-Genome Sequencing (WGS)
7 Conclusion
References
Chapter 9: Generation of Knockout and Fragment Deletion Mutants in Soybean by CRISPR-Cas9
1 Introduction
2 Materials
2.1 Soybean Cultivar
2.2 Vector and Agrobacterium Strain
2.3 Agrobacterium Strain Preparation and Soybean Transformation
3 Methods
3.1 Design and Selection of sgRNAs (See Fig. 1a)
3.2 Construction of the CRISPR-Cas9 Vector for Gene Knockout (See Fig. 1b)
3.3 Construction of the CRISPR-Cas9 Vector for Large Fragment Deletions (See Fig. 1c)
3.4 Preparation of Agrobacterium tumefaciens
3.5 Generation of Transgenic Soybean Plants by Using Agrobacterium-Mediated Transformation
3.6 Identification of T0 Mutants
3.7 Identification of the Heritable Mutations from Progeny
3.8 Identification of ``Transgene-Free´´ Mutants
4 Notes
References
Chapter 10: Targeted Base Editing in Soybean Using a CRISPR-Cas9 Cytidine Deaminase Fusion
1 Introduction
2 Materials
2.1 Soybean Cultivar
2.2 Vector and Agrobacterium Strain
2.3 Agrobacterium Strain Preparation and Soybean Transformation
3 Methods
3.1 Designing and Selection of sgRNA (See Fig. 1a)
3.2 Construction of Vector for Base Editing (See Fig. 1b)
3.3 Preparation of Agrobacterium tumefaciens
3.4 Generating Transgenic Soybean Plants by Agrobacterium-Mediated Transformation
3.5 Identifying Mutants
4 Notes
References
Chapter 11: Efficient Genome Editing in Potato Using a Hairy Root Transformation System
1 Introduction
2 Materials
2.1 Plant Tissue Culture
2.2 Bacterial Culture
3 Methods
3.1 Preparation of Potato Explants
3.2 Kill Curve Experiment
3.3 Preparing A. rhizogenes Infection Solution
3.4 Inoculation and Co-culture of Potato Explants with A. rhizogenes
3.5 Sub-Culturing and Sampling Transgenic Hairy Root Clones
3.6 Regenerating Transgenic Hairy Root Clones
4 Notes
References
Chapter 12: Efficient CRISPR-Cas9-Mediated Genome Editing in Tomato
1 Introduction
2 Materials
2.1 Agrobacterium tumefaciens Strain
2.2 Plant Material
2.3 Stock Solutions
2.4 Culture Media (1 L)
2.5 Other Reagents (Molecular Biology and Cloning)
3 Methods
3.1 Selection and Design of Single Guide RNAs
3.2 Design of Vector Construct for Single Mutations
3.3 Design of Vector Construct for Double Mutations
3.3.1 First Approach
3.3.2 Second Approach
3.3.3 Third Approach
3.4 Transformation and Tissue Culture
3.4.1 Seed Preparation and Germination
3.4.2 Agrobacterium Culture and Co-cultivation
3.4.3 Pre-cultivation
3.4.4 Co-cultivation
3.4.5 Initiation of Calli and Regeneration of Shoot
3.4.6 Differentiation of Transgenic Shoots
3.4.7 Rooting of Transgenic Shoots
3.4.8 Transplanting and Acclimation
3.4.9 High-Throughput Identification of Mutations
Construction of NGS Library
Next-Generation Sequencing
4 Notes
References
Chapter 13: CRISPR-Cas9-Mediated Genome Editing in Rice: A Systematic Protocol for Single- and Multi-Target Vector Construction
1 Introduction
2 Materials
2.1 Vector
2.2 Reagents
2.3 Organisms
2.4 Antibiotic Stock Solution
2.5 Liquid Culture Media
2.6 Solid Culture Media
3 Methods
3.1 Target Selection and Primer Design
3.1.1 Target Site Selection
3.1.2 Design Two Complementary DNA Sequences
3.2 Intermediate Vector Construction
3.2.1 Culture of E. coli Cells Harboring Sk-gRNA Vector
3.2.2 A Single Digestion of SK-gRNA
3.2.3 Gel Electrophoresis and Purification
3.2.4 Preparation of Guide Sequence Primer
3.2.5 A Ligation Reaction System
3.2.6 Transformation of the Constructed Plasmid into Competent DH5α Cells
3.2.7 Bacterial Colony Culture
3.2.8 Bacterial Colony PCR
3.2.9 PCR Cycling Process
3.2.10 Gel Electrophoresis
3.2.11 Sequencing of Positive Colony
3.3 Final Vector Construction
3.3.1 Culture of E. coli Cells Harboring pC1300-Cas9 Vector
3.3.2 Culture of Recombinant SK-gRNA
3.3.3 A Double Digestion of pC1300-Cas9
3.3.4 Gel Electrophoresis and Purification
3.3.5 A Double Digestion of Recombinant SK-gRNA
3.3.6 Gel Electrophoresis and Purification
3.3.7 Ligation Reaction System
3.3.8 Transformation of the Constructed Plasmid into Competent DH5α Cells
3.3.9 Bacterial Colony Culture
3.3.10 Bacterial Colony PCR
3.3.11 PCR Cycling Process
3.3.12 Gel Electrophoresis
3.3.13 Sequencing of Positive Colony
3.4 Vector Construction for Two Target Sites Following Two-Step Methods
3.4.1 Culture of Recombinant SK-gRNA
3.4.2 A Double Digestion of Recombinant SK-gRNA Having Guide Sequence 1
3.4.3 Gel Electrophoresis and Purification
3.4.4 A Double Digestion of Recombinant SK-gRNA Having Guide Sequence 2
3.4.5 Gel Electrophoresis and Purification
3.4.6 Ligation Reaction System
3.4.7 Transformation of the Constructed Plasmid into Competent DH5α Cells
3.4.8 Bacterial Colony Culture
3.4.9 Bacterial Colony PCR
3.4.10 PCR Cycling Process
3.4.11 Gel Electrophoresis
3.4.12 Sequencing of Positive Colony
3.4.13 Culture of E. coli Cells Harboring pC1300-Cas9 Vector
3.4.14 Culture of Recombinant SK-gRNA
3.4.15 A Double Digestion of pC1300-Cas9
3.4.16 Gel Electrophoresis and Purification
3.4.17 A Double Digestion of Recombinant SK-gRNA Having Guide Sequence 1 and 2
3.4.18 Gel Electrophoresis and Purification
3.4.19 Ligation Reaction System
3.4.20 Transformation of the Constructed Plasmid into Competent DH5α Cells
3.4.21 Bacterial Colony Culture
3.4.22 Bacteria Colony PCR
3.4.23 PCR Cycling Process
3.4.24 Gel Electrophoresis
3.4.25 Sequencing of Positive Colony
3.5 Vector Construction for Two Target Sites Following One-Step Methods
3.5.1 Culture of E. coli Cells Harboring pC1300-Cas9 Vector
3.5.2 Culture of Recombinant SK-gRNA
3.5.3 A Double Digestion of pC1300-Cas9
3.5.4 Gel Electrophoresis and Purification
3.5.5 A Double Digestion of Recombinant SK-gRNA Having Guide Sequence 2
3.5.6 Gel Electrophoresis and Purification
3.5.7 A Double Digestion of Recombinant SK-gRNA Having Guide Sequence 1
3.5.8 Gel Electrophoresis and Purification
3.5.9 Ligation Reaction System
3.5.10 Transformation of the Constructed Plasmid into Competent DH5α Cells
3.5.11 Bacterial Colony Culture
3.5.12 Bacteria Colony PCR
3.5.13 PCR Cycling Process
3.5.14 Gel Electrophoresis
3.5.15 Sequencing of Positive Colony
3.6 Vector Construction for Three Target Sites
3.7 Vector Construction for Four Target Sites
3.8 Agrobacterium-Mediated Transformation in Rice
3.9 Mutation Detection and Analysis of Transgenic Plants
References
Chapter 14: Generating Clonal Seeds from Hybrid Rice with CRISPR-Cas9
1 Introduction
2 Materials
2.1 Plant
2.2 Plasmids
2.3 Bacterial Strain and Growth Medium
2.4 Chemicals, Buffers, and Solutions
2.5 Medium Used in Rice Transformation
3 Methods
3.1 Primer Design
3.2 Vector Construction
3.3 Rice Transformation and Detection of Genome Modifications
3.4 Identification of Clonal Plants
4 Notes
References
Chapter 15: CRISPR-Cas12a-Based DNA Detection for Fast Pathogen Diagnosis and GMO Test in Plants
1 Introduction
2 Materials
2.1 RNA Synthesis and Purification
2.2 Filter Paper-Based DNA Isolation
2.3 RPA Amplification
2.4 Lateral Flow Assay
3 Methods
3.1 Design of RPA Primer and crRNA
3.1.1 Select Target Gene for Detection
3.2 crRNA Synthesis
3.2.1 In Vitro Transcription of crRNA
3.2.2 Purification of crRNA
3.2.3 Examine the Quality and Activity of crRNA (Optional)
3.3 All-Paper-Based Gene Detection Using Cas12a
4 Notes
References
Chapter 16: CRISPR-Cas9-Mediated Gene Editing in Human Induced Pluripotent Stem Cells
1 Introduction
2 Generation of the Targeting Plasmid
2.1 Materials
2.2 Procedure
2.2.1 Annealing of the Complementary Oligonucleotides to Form an Oligonucleotide Duplex
2.2.2 Digestion of the pCRII-BbsI-sgRNA Scaffold Plasmid
2.2.3 Ligation of the Oligonucleotide Duplex into the pCRII-BbsI-sgRNA Scaffold Plasmid
3 Generation of the Donor Construct
3.1 Materials
3.2 Procedure
3.2.1 Digestion of the EF1a-cDNA-pCAG-Neo Plasmid
3.2.2 Ligation of the cDNA Insert into the EF1a-cDNA-pCAG-Neo Plasmid
4 Generation of Conditioned Media
4.1 Materials
4.2 Procedure
5 Preparation of the DNA Prep (1 Day in Advance)
5.1 Materials
5.2 Procedure
6 Plating MEFs
6.1 Materials
6.2 Procedure
7 Nucleofection of hiPSCs
7.1 Materials
7.2 Procedure
8 Picking Colonies
8.1 Materials
8.2 Procedure
9 Genotyping
9.1 DNA Isolation
9.1.1 Materials
9.1.2 Procedure
9.2 Genotyping Method 1: Introduction of Indels (Nested DNA Sequencing)
9.2.1 Materials
9.2.2 Procedure
9.2.3 Results
9.3 Genotyping Method 2: Introduction of Large Deletions
9.3.1 Materials
9.3.2 Procedure
9.3.3 Results
9.4 Genotyping Method 3: Knock-In with Donor Template Integration
9.4.1 Materials
9.4.2 Procedure
9.4.3 Results
10 Protocol Adjustments for Feeder-Free hiPSCs
10.1 Generation of Conditioned Media
10.2 Plating Frozen MEFs
10.3 Nucleofection of hiPSCs
10.3.1 Materials
10.3.2 Procedure
10.4 Picking Colonies
10.4.1 Materials
10.4.2 Procedure
11 Troubleshooting
12 Notes
References
Chapter 17: Cell-Type-Specific CRISPR-Cas9 System with miRNAs
Abbreviations
1 Introduction
2 Materials
2.1 RNA Template and RNA Construction
2.2 Cell Culture, RNA Transfection and Assay
3 Methods
3.1 Construction of the miRNA-Responsive Cas9/AcrllA4 mRNA Template and the sgRNA Template (Fig. 2)
3.1.1 Choosing the Target miRNA and Constructing the Control 5′-UTR by PCR
3.1.2 Construction of the Cas9/AcrllA4 Coding Region by PCR
3.1.3 Construction of the 3′-UTR by PCR
3.1.4 Construction of the Full-Length mRNA Template by Fusion PCR
3.1.5 Construction of the sgRNA Template by PCR
3.2 Construction of RNA by IVT (See Note 2)
3.2.1 Generating and Purifying mRNA
3.2.2 Generating and Purifying sgRNA
3.3 Validation
3.3.1 Preparation of Samples
3.3.2 Transfection of OFF System into HeLa-EGFP
3.3.3 Transfection of ON System into HeLa-EGFP
3.3.4 Validation of Genome-Editing Efficiency
4 Notes
References
Chapter 18: CRISPR/Cas9 Gene Editing in Mammalian Cells Using LentiCRISPRv2/LentiGuide-Puro Vectors
1 Introduction
1.1 CRISPR Cas9
1.1.1 Lentivirus Vector Design
2 Materials
3 Methods
3.1 Design of Oligos
3.2 Digestion of Vector Using BsmBl Restriction Enzyme
3.2.1 Procedure for Vector Digestion
3.3 Perform Gel Electrophoresis to Check for Vector Digestion
3.4 Procedure for DNA Extraction
3.5 Annealing and Phosphorylation of Oligos
3.5.1 Procedure
3.6 Gel Electrophoresis to Check for Annealing
3.7 Ligation Reaction
3.7.1 Procedure for Ligation
3.8 Carry out Restriction Endonuclease Digestion and Run on a Gel to Check for Successful Ligation
3.9 Transformation
3.10 Picking of Colonies
3.11 Plasmid Isolation
3.12 Restriction Endonuclease Reactions to Check Transformation Products
3.13 Lentiviral Packaging (Transfection of HEK293T Cells)
3.14 Transduction of MeT-5A-Cas9 Cells
3.15 DNA Isolation
3.16 T7 Endonuclease I (T7E1) Assay
3.16.1 PCR Amplification
3.16.2 Gel Extraction
3.16.3 T7E1 Assay
4 Notes
References
Chapter 19: An Approach to Proximity Ligation by T4 RNA Ligase to Screen sRNA That Regulate CRISPR-Cas Systems
1 Introduction
2 Materials
2.1 Plasmid Vectors Preparations
2.2 Bacterial Strains
2.3 Oligonucleotides
2.4 Reagents
3 Methods
3.1 Preparation of RNA Ligation for sRNA-CRISPR Leader Chimeras in the Cells
3.2 RNA Isolation and cDNA Synthesis
3.3 Detection of sRNA-CRISPR Leader Chimeras by RT-PCR
4 Notes
References
Chapter 20: Genetic Engineering of a Phage-Based Delivery System for Endogenous III-A CRISPR-Cas System Against Mycobacterium ...
1 Introduction
2 Materials
2.1 Strains and Vectors
2.2 E. coli, M. smegmatis, and M. tuberculosis Growth Media
2.3 Reagents for Vector Construction
3 Methods
3.1 Cloning of Artificial Mini-CRISPR Plasmid
3.1.1 Selection of Target Sequences
3.1.2 Construction of Mini-CRISPR Plasmid
3.2 Construction of Engineered Phages
3.2.1 Construction of phAE159 Vector
3.2.2 Transform the Confirmed Recombinant phAE159 to M. smegmatis mc2155
3.2.3 Collection of Engineered Phages
3.3 Transformation and Infection of M. tuberculosis H37Ra
3.3.1 Transform Recombinant pMV26-Mini-CRISPR Plasmids to M. tuberculosis H37Ra
3.3.2 Infection of M. tuberculosis H37Ra with Engineered Phages
4 Conclusions
5 Notes
References
Chapter 21: CRISPR-Cas9-Mediated Genome Editing in Escherichia coli Bacteriophages
1 Introduction
2 Materials
2.1 Strains and Vectors
2.2 E. coli Growth Media
2.3 Reagents for Vector Construction
2.4 Media and Supplies for E. coli Transformation
2.5 Materials for Detection and Isolation of Mutated Phages
3 Methods
3.1 Selection of Target Sequences
3.2 Construction of Genome Editing Plasmids (See Note 1)
3.2.1 Construction of gRNA Plasmid
3.2.2 Construction of Homologous Donor Plasmids
3.3 Transformation of E. coli BL21 (DE3)
3.4 Phage Infecting E. coli
3.5 Isolation of Phage Mutants
3.6 Detection and Examination of the Genotype of Phage Mutants (Fig. 2f)
3.6.1 Detection of Point Mutation of Target Gene
3.6.2 Detection of the Genotype of the Inserted Reporter Gene
4 Notes
References
Chapter 22: CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum
1 Introduction
2 Materials
2.1 Culture Media
2.2 Cloning and Transformation
3 Methods
3.1 gRNA Design
3.2 Homology Arm Design
3.3 Plasmid Construction
3.4 Transformation into C. thermocellum
4 Notes
References
Chapter 23: Gene Targeting in Caenorhabditis elegans Using a Combination of Multiple sgRNAs and a Homologous Recombination-Med...
1 Introduction
2 Materials
2.1 Software Tools and Databases
2.2 Nematode Growth Medium
2.3 Plasmids
2.4 Reagents
2.5 DNA Isolation
2.6 Animals
2.7 Transgenesis
2.8 Worm Screening
3 Methods
3.1 Worm Culture and Maintenance
3.2 Single Worm DNA Isolation
3.3 sgRNA and Homology Arms Design
3.4 Cloning of sgRNA Through Golden Gate Assembly
3.5 Cloning of Repair Template Through Gibson Cloning Assembly
3.6 Transgenesis
3.7 Mutant Screening
4 Notes
References
Chapter 24: Design of Repair Templates for CRISPR-Cas9-Triggered Homologous Recombination in Caenorhabditis elegans
1 Introduction
1.1 ZFN, TALEN, and CRISPR-Cas
1.2 C. elegans and CRISPR-Cas
2 Materials
2.1 Reagents
2.2 Software
2.3 Primer Sequences
3 Methods
3.1 Single-Strand Oligonucleotides as Repair Templates
3.1.1 Procedure
3.2 Screening for Substitution
3.3 Screening for Insertion or Deletion
3.3.1 Insertion
3.3.2 Deletion
3.4 Linear Double-Stranded DNA (dsDNA)
3.4.1 Procedure
3.5 Plasmid as a Repair Template
3.6 Prebuilt Repair Template Plasmids for CRISPR-Cas9
3.7 Building a Custom Repair Template Plasmid
3.7.1 Procedure
4 Notes
References
Correction to: CRISPR-Cas9-Mediated Genome Editing in Rice: A Systematic Protocol for Single- and Multi-Target Vector Construc
Correction to: CRISPR-Cas9-mediated Gene Editing in Human Induced Pluripotent Stem Cells
Glossary
References
Index