Lecture Notes: Genome Editing and Engineering - Targeted Genetic Modification

Introduction

• Topic: Targeted Genetic Modification (Module 4, Part 2)
• Focus: Knockouts and Knock-ins

Gene Modification Techniques

Knockouts

• Definition: Technique that makes genes non-functional.
• Purpose: Study gene function by comparing knockout mutants with wild type.
• Key Points:
  • Knockouts = loss of gene function.
  • Useful for studying gene function differences.

Knock-ins

• Definition: Technique that provides gain of gene function.
• Purpose: Similar to knockouts, allows study of gene functions by comparing knock-in mutants with wild types.
• Key Points:
  • Can create multiple knockouts/knock-ins in the same organism.
  • Generates double knockouts (DKO), triple knockouts (TKO), etc.

Creating Knockouts and Knock-ins

• Types of Knockouts:

  • Heterozygous Knockout: One allele inactive, one wild type.
  • Homozygous Knockout: Both alleles inactive.

• Vectors in Knockouts:

  • Contain:
    • Stretch of nucleotides homologous to the target gene.
    • Selection markers for successful knockouts.
    • Restriction sites for linearization to facilitate homologous recombination.

Homologous Arms

• Definition: DNA stretches that flank the target gene.
• Length Requirement: About 2 kb minimum; typically 6 to 14 kb used.
• Negative and Positive Selection Markers:
  • Positive markers (e.g., neomycin, puromycin) used for successful knockout selection.
  • Negative markers (e.g., HSV-TK) kill cells with random integrations.

Types of Vectors

Replacement Vectors

• Description: Replace the target gene with a selection marker gene.
• Key Features:
  • Two homology arms flanking the selection marker.
  • Used primarily for generating knockout mice.

Insertion Vectors

• Description: Insert a sequence into the target gene, disrupting it.
• Key Features:
  • One homologous arm with a drug selection gene.
  • Mainly used for point mutations via a hit-and-run method.

Techniques for Subtle Mutations

• Methods:
  1. Hit and Run Approach
  2. Tag and Exchange Approach
  3. Recombination Based Approach

Hit and Run Approach Steps:

1. Use an insertion vector for homologous recombination to introduce a mutation.
2. Intrachromosomal recombination leads to excision of selection genes, retaining the mutation.

Tag and Exchange Approach Steps:

1. First homologous recombination replaces exon with a selection marker.
2. Second targeting event replaces the selection marker with a point mutation.

Designing Knock-out Constructs

Steps:

1. Retrieve DNA Sequence: For the gene of interest (15 kb upstream and downstream).
2. Design Primers for Homology Arms: Ideal length and Tm considerations.
3. Genomic DNA Isolation: Use standard protocols.
4. Assembly of Homology Arms and Selection Markers: Utilize ligation and cloning techniques.

Key Considerations for Primers:

• Length: 23-30 base pairs.
• Annealing Temperature: 60-68°C.
• GC content: 40-60%.

Transformation Techniques

• Microinjection: High efficiency but tedious.
• Electroporation: Mass delivery system for targeting vectors.

Verification of Targeting

Southern Blotting

• Identifies homologous recombinant clones.
• Bands indicate presence of wild type and targeted alleles.

Humanization of Experimental Models

• Knocking out mouse genes not similar to humans and replacing with human genes.
• Goal: Create more accurate mouse models for studying human diseases.

Example: CD14 Gene Knock-in Strategy

• Targeting vector integrates human CD89 into the mouse genome.
• Result: Transgenic mice expressing human genes.

Conclusion

• Summary of knockout and knock-in production processes.
• Emphasis on the relevance of these techniques in genetic research.
• Thank you for your attention!