Genomic Techniques (Gene Editing)

History of Genome Editing

Key Scientific Milestones

1. Identification of DNA as Genetic Material: The discovery that DNA, not protein, is the genetic material in cells.
2. Structure of DNA: James Watson and Francis Crick described the double helix structure of DNA.
3. Discovery of Restriction Enzymes: Enzymes that cut DNA at specific sequences, paving the way for genetic engineering.
4. Creation of Recombinant DNA: The first successful splicing of DNA from different organisms.
5. PCR (Polymerase Chain Reaction): A technique developed to amplify DNA sequences, essential for genetic analysis and manipulation.

Evolution of Genetic Manipulation Methods

1. Basic Techniques:

   • Cloning and Recombinant DNA Technology: Early methods of copying and combining genetic material.
   • Transgenic Models: Creating organisms that carry genes from other species.

2. Advanced Genome Editing:

   • Zinc Finger Nucleases (ZFNs): Engineered proteins that create double-strand breaks at specific locations in the DNA.
   • TALENs (Transcription Activator-Like Effector Nucleases): Like ZFNs, but easier to design for specific targets.
   • CRISPR-Cas9: A revolutionary technique that simplified and democratized genome editing. It uses a guide RNA to direct Cas9 nuclease to a specific sequence in the genome, where it creates a double-strand break.

Types of Genome Engineering

Meganucleases

Description and Mechanism of Action

• Meganucleases are a group of endonucleases that recognize and cut large DNA sequences (12-40 base pairs).
• They work by creating double-strand breaks in DNA at specific sites, which the cell then repairs, allowing for genetic modifications.

Historical Significance and Applications

• Historically significant as one of the earliest tools for genome editing.
• Used in gene therapy, functional genomics, and the development of transgenic plants and animals.

ZFN (Zinc Finger Nucleases)

Structure and Function

• ZFNs consist of a zinc finger DNA-binding domain fused to a DNA-cleavage domain.
• They recognize specific DNA sequences through the zinc finger domain and create double-strand breaks in DNA.

Methodology of Genome Editing with ZFN

• Two ZFN molecules bind to their target DNA sequence, bringing the cleavage domains together to cut the DNA.
• This break is then repaired by the cell’s machinery, leading to targeted genetic modifications.

Limitations

• Long synthesis time and nonmodular assembly process
• Not for every genomic locus

TALENs (Transcription Activator-Like Effector Nucleases)

Composition and Working Principle

• TALENs are similar to ZFNs but use a different DNA-binding domain derived from TAL effectors of Xanthomonas bacteria.
• They bind to specific DNA sequences and induce double-strand breaks for targeted genome editing.

Comparison with Other Genome Editing Techniques

• More versatile and easier to design than ZFNs due to their modular DNA-binding domain.
• However, CRISPR-Cas9 has largely surpassed TALENs in popularity due to its simplicity and efficiency.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

Origin and Discovery of CRISPR

• Discovered as a part of the bacterial immune system, where it provides a defense mechanism against viruses.
• Comprises short repetitions of base sequences, interspaced with spacer DNA from previous viral exposures.

CRISPR-Cas9 System and its Role in Genome Editing

• CRISPR-Cas9 revolutionized genome editing. It uses a guide RNA to direct the Cas9 nuclease to a specific DNA sequence.
• It allows for easy, precise, and efficient modification of genes in a wide range of organisms.
• Its versatility and ease of use have made it the preferred tool for many genome editing applications.

Feature/Technology	Meganucleases	ZFNs (Zinc Finger Nucleases)	TALENs (Transcription Activator-Like Effector Nucleases)	CRISPR/Cas9
Recognition Sequence	14-40 bp	9-18 bp	15-20 bp per TALE unit	20 bp guide RNA + PAM
Efficiency	Moderate	High	High	Very High
Specificity	High	Moderate	High	High
Off-Target Effects	Low	Higher than TALENs	Lower than ZFNs	Moderate, depends on guide RNA design
Customizability	Low	Moderate	High	Very High
Complexity	High	High	Moderate	Low
Flexibility	Low	Moderate	High	Very High
Development Time	Long	Long	Moderate	Short
Cost	High	High	Moderate	Relatively Low
Typical Applications	Research	Gene therapy, research	Gene therapy, research	Gene editing, research, therapeutics

Current and Novel Genome Engineering Mechanisms

Mechanisms of Action

• Detailed understanding of how each genome editing technique modifies genetic material.

Comparative Analysis

• Strengths, limitations, and potential applications of each method.

CRISPR-Cas9 Delivery in Eukaryotes

CRISPR Applications

Gene-knockout, gene-knockin, gene tagging, base editing, prime editting…

CRISPR-inhibitor: Silences gene

CRISPR-activator: Expresses gene

Anti-CRISPR proteins: Inhibits CRISPR activity, reducing off-target effects.

Gene tagging: Adding a fluorescent protein to a gene of interest to track its expression and localization.

CRISPR experiment

1. Design: Design guide RNA and choose an appropriate Cas nuclease.

Tools: benchling, e-crisp

2. Edit: Optimize the delivery method for the CRISPR components.

   • Viral Vectors: Adenoviruses, AAVs, Lentiviruses.
   • Electroporation: Electric pulses to create temporary pores in cell membranes.
   • Cell-penetrating Peptides: Short peptides that can transport CRISPR components into cells.
   • Nanoparticles: Lipids, Liposomes, Polymeric and inorganic nanoparticles.

3. Analyze: Assess the efficacy and specificity of the genome editing.

Tools: Sanger sequencing, synthego, tide

Examples of CRISPR Applications

Mutation correction using CRISPR/Cas9.

Ongoing debates

• Germline Editing: The use of CRISPR in human embryos, eggs, or sperm is controversial due to ethical concerns and the potential for heritable changes.