Gene Editing

  1. The CRISPR-Cas9 System in Detail
    1. Core Components
      1. Cas9 Protein Structure and Function
        1. Overall Architecture
          1. Functional Domains
            1. Recognition Lobe
              1. Nuclease Lobe
                1. Bridge Helix
                2. Nuclease Domains
                  1. HNH Domain
                    1. RuvC Domain
                    2. DNA Binding and Cleavage
                      1. Conformational Changes
                        1. Cleavage Mechanism
                      2. Guide RNA Components
                        1. CRISPR RNA
                          1. Spacer Sequence
                            1. Repeat Sequence
                            2. trans-activating crRNA
                              1. Secondary Structure
                                1. Cas9 Interaction
                                2. Single-guide RNA Design
                                  1. Fusion of crRNA and tracrRNA
                                    1. Optimization Strategies
                                      1. Length Considerations
                                  2. Mechanism of Action
                                    1. Target Recognition Process
                                      1. PAM Sequence Recognition
                                        1. PAM Requirements
                                          1. PAM Variants
                                          2. R-loop Formation
                                            1. DNA Unwinding
                                              1. RNA-DNA Hybridization
                                              2. Conformational Activation
                                              3. DNA Cleavage
                                                1. Double-Strand Break Formation
                                                  1. Cleavage Site Positioning
                                                    1. Blunt-end Generation
                                                    2. Factors Affecting Efficiency
                                                      1. Guide RNA Design
                                                        1. Chromatin Accessibility
                                                          1. Target Site Context
                                                        2. Cellular DNA Repair Outcomes
                                                          1. Non-Homologous End Joining
                                                            1. NHEJ Pathway Components
                                                              1. Repair Outcomes
                                                                1. Perfect Repair
                                                                  1. Insertions and Deletions
                                                                  2. Applications in Gene Knockout
                                                                    1. Frameshift Generation
                                                                      1. Functional Disruption
                                                                    2. Homology-Directed Repair
                                                                      1. HDR Pathway Requirements
                                                                        1. Donor Template Design
                                                                          1. Single-stranded Oligonucleotides
                                                                            1. Double-stranded DNA Templates
                                                                              1. Homology Arm Length
                                                                              2. Applications
                                                                                1. Gene Correction
                                                                                  1. Precise Insertions
                                                                                    1. Reporter Integration
                                                                                    2. Factors Affecting HDR Efficiency
                                                                                      1. Cell Cycle Dependence
                                                                                        1. Template Concentration
                                                                                          1. Delivery Methods
                                                                                        2. Alternative Repair Pathways
                                                                                          1. Microhomology-Mediated End Joining
                                                                                            1. Mechanism
                                                                                              1. Predictable Deletions
                                                                                              2. Single-Strand Annealing
                                                                                                1. Break-Induced Replication

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                                                                                            4. The CRISPR-Cas System

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                                                                                            6. Variations and Innovations in CRISPR Technology