Gene editing

Question 1

Why edit genomes?

Answer 1

• Model human genetic disease in animal models, study human pathways
• Correct pathogenic mutations in cell lines for therapy and personalised medicine e.g blood disorders or autoimmune diseases (personalised medicine)
• Improve key organisms for biotechnology e.g. plants, livestock, yeast and bacterial strains

Question 2

DSB associated with HDR

Answer 2

The Problem: Double-Strand Break (DSB)
The most dangerous type of DNA damage where the helix is snapped in two.
The Preparation: Resection
Enzymes trim the 5’ ends to create 3’ single-stranded DNA tails. These tails are “homology seekers.”
The Search: Strand Invasion & D-Loop
The 3’ tail invades a matching (blue) sister chromatid. This creates a Displacement Loop (D-Loop), using the intact DNA as a perfect template for new synthesis.
The Two Fixes (Pathways):
* SDSA (Synthesis-Dependent Strand Annealing): The new strand is kicked off the template and “anneals” back to its original partner. This is the “clean” repair used in normal cell division (Non-crossover).
* DSBR (Double-Strand Break Repair): Both ends are captured, creating Double Holliday Junctions. This can result in a Crossover (DNA swapping), which is the engine for genetic diversity during meiosis.

Question 3

Classical Double Stranded Break Repair (DSBR)

Answer 3

• DSB: DNA damaging agents
• Resection: nuclease degradation, SS 3’ tails. RPA
• Homology Searching: RAD51
• D-Loop: invading strand forms a loop, acts as a primer for DNA synthesis.
• Holliday Junction: intercrossing DNA, during recombination.

Question 4

Synthesis-Dependent Strand-Annealing Pathway

Answer 4

• No cross over events!!
• Newly synthesised strand displaced from template
  and returns to the processed end of non invading strand.

Question 5

Non Homologous End-Joining (NHEJ)

Answer 5

DSB caused by DNA damaging agents is random….although there are hot spots!

Question 6

Meganucleases (MNs)

Answer 6

• Endodeoxyribonucleases, most specific naturally occurring REs
• Can tolerate 1 or 2 MMs. (3x and 107x /human genome).
• Examples: I-SceI (baker’s yeast), I-CreI (green algae), I DmoI (Archaebacteria)
• I-SceI, 18 bp Rec. Site. Predicted to recognise identical seq once for a genome 20x size of human genome.
• Can insert DNA by HR or mutations by NHEJ
• Difficult to specifically target to roi! Chimeric MNs…?

Question 7

What do we want from an endonuclease?

Answer 7

• Specific recognition of long target sequences (ideally one per genome)
• Adaptability for retargeting to other genomic loci

Question 8

Zinc Finger Nucleases (ZFNs)

Answer 8

• ZFs are DNA binding domains
• Each finger (30aa) recognises 3 bps
• An array of zf domains : recognises unique genomic sequences

• ZF are fused to FokI
• FokI needs to dimerise to cut: ZFN come as pairs (specificity!)
• Complex, expensive, can have inaccurate cleavage (domain interaction)

Question 9

Transcription Activator-Like Effector Nucleases (TALENs)

Answer 9

• TALE proteins: Xanthomonas, plant pathogenic bacteria
• DNA binding domains: series of tandem repeats (33-35 aa)
• Variations at aa 12 + 13 confers specificity to nucleotides (1 repeat:1 bp)
• Much easier to design than ZFN, fewer targeting constraints.
• Much larger than ZFNs (3kb Vs 1Kb), harder to deliver.
• Off-target a concern, WGS shows it’s low.

Question 10

CRISPR/Cas9 System

Answer 10

• Clustered regularly interspaced short palindromic repeats (CRISPR)
• CRISPR associated protein 9 (Cas9) - Bacterial adaptive immune system
• Protospacer (Target sequence of guide RNA)
• Protospacer Adjacent Motif (PAM)
• Guide RNA binds to strand of genomic DNA, Cas9 endonuclease binds to non protospacer portion of gRNA + PAM of DNA, DSB 3bp upstream of PAM

Question 11

Comparison of different techniques

Answer 11

Question 12

Duchenne Muscular Dystrophy (DMD)

Answer 12

• DMD most common severe form of childhood muscular dystrophy (1:5000 males). Mutated gene: Dystrophin
• Skeletal + cardiac
• Unable to walk by 10- 12 yrs, Death by early to mid
• 20s (heart failure)
• Current treatments: Corticosteroids (side effects!), morpholino based exon skipping (prolonged ambulation).
• Gene therapy difficult, dystrophin 11kb (adeno-associated virus [AAV])

Question 13

Strategy

Answer 13

The Problem: Nonsense Mutation
The top-right diagram shows a gene where Exon 23 (in red) contains a mutation represented by the red lollipop. This is a “nonsense” mutation, meaning it creates a premature stop signal that prevents the protein from being finished.
The Tool: Dual gRNA and Cas9
In the center, you see the Cas9 protein (the large yellow blob) holding a guide RNA (gRNA).
* The Strategy: Instead of trying to fix the tiny mutation, scientists use two gRNAs (gRNA1 and gRNA2) to target the DNA flanking Exon 23.
* The Cut: Cas9 acts like molecular scissors to create two Double-Strand Breaks (DSB), essentially “cutting out” the entire faulty exon.
The Repair: Non-Homologous End Joining (NHEJ)
Once the DNA is cut, the cell’s natural repair mechanism, NHEJ, takes over.
* The Result: The cell “stitches” Exon 22 directly to Exon 24.
* Exon Skipping: Because Exon 23 is gone, the “stop” signal is removed. This allows the rest of the protein (Exon 24, 25, etc.) to be produced. The resulting protein is slightly shorter but still functional.

The Biological Proof (The Mice)
On the left, you see two mice. This type of research often shows a “before and after” effect:
* The untreated mouse suffers from muscle wasting.
* The treated mouse, having had the mutation “skipped” via CRISPR, shows improved muscle mass and strength.

Question 14

Transfection of constructs into dystrophic satellite cells (in vitro)

Answer 14

The Strategy (Top Diagrams)
* Panel A (Ai9 Reporter): To see if the CRISPR machinery is working, scientists used a “stoplight” system. They inserted a STOP signal between a green promoter and a red tdTomato gene. When CRISPR cuts out the STOP signal, the cells turn red.
* Panel C (Dmd Gene Editing): This shows the actual therapeutic target. Two gRNAs cut out Exon 23 (which contains the harmful mutation). By removing this exon, the cell “skips” the error and connects Exon 22 directly to Exon 24, allowing a functional Dystrophin protein to be made.
Molecular Evidence (Bottom Left Gels)
These gels act as a “DNA/RNA fingerprint” to prove the editing happened:
* Gel A (Genomic DNA): Look for the red asterisk (∗). The smaller band in the “Ai9+Dmd gRNAs” group shows that a piece of DNA was successfully deleted.
* Gel B (mRNA): Look for the blue asterisk (∗). This shows the mRNA is shorter because Exon 23 has been removed (Exon Skipping).
* Gel C (Western Blot): This proves the protein is actually being made. The rows labeled DYS show bands appearing only when the CRISPR gRNAs are used, indicating that the cells are now successfully producing the Dystrophin protein.

Visual Proof (Bottom Right Microscopy)
This is the “Before and After” look at the cells:
* Top Row (Control): The cells have no Dystrophin (black square) and very few are red.
* Bottom Row (Edited): * Dystrophin (Green): You see a clear “honeycomb” pattern. This is the Dystrophin protein correctly sitting on the membrane of the muscle cells.

* tdTomato (Red): Many cells are glowing red, proving the CRISPR machinery successfully "cut out" the stop signals.
* Merge: The final image shows the overlap of the red (edited cells) and green (recovered protein).

Question 15

AAV-CRISPR excises E23 + restores Dystrophin expression (in vivo)

Answer 15

Genomic and Molecular Restoration
* DNA Correction (Panel A): Using specific guide RNAs (gRNAs) targeting the Dmd gene, the researchers successfully edited the Genomic DNA. The red asterisk indicates the presence of the edited DNA band.
* mRNA Expression (Panel B): The editing resulted in the production of modified mRNA transcripts (blue asterisk), which are necessary for protein synthesis.
* Protein Expression (Panels C & D): * Western Blot (C): Shows that Dystrophin (DYS) protein, which is completely absent in the mdx (DMD) mouse model, is restored following treatment with Dmd gRNAs.
* Immunofluorescence (D): Visually confirms the presence of Dystrophin (green) localized at the muscle fiber membranes in treated mice, compared to the empty blue (DAPI/nuclei only) fields in the control.
Functional Recovery (Panels E & F)
The goal of the therapy is to restore muscle strength. These graphs compare Wild type (healthy), treated mice, and control mice:
* Specific Force (E): Measures the strength of the muscle. The treated groups (AAV-Dmd CRISPR) show a significant increase in force compared to the untreated mdx baseline.
* Force Drop (F): Measures how much strength the muscle loses after eccentric contractions (a sign of muscle fragility). Treated mice show better resistance to this damage.
Experimental Setup and Clinical Outlook
* Surgical Models: The right-hand diagrams show the physiological testing setup, including the tibial nerve stimulation and the micromanipulator used to measure the force generated by the mouse’s leg muscles.
* Clinical Potential: The text “Proof of principle…clinical trials?” suggests that since this worked in mice, the next logical step is moving toward human therapies to fix the genetic mutations causing DMD.

Question 16

Case Study 2: MYBPC3

Answer 16

• The Mutation: A deletion in the MYBPC3 gene, which causes a common form of inherited heart disease.
• Technical Breakthrough: By injecting CRISPR/Cas9 components directly into the oocyte at the time of fertilization (Sperm + CRISPR), researchers successfully reduced Mosaicism.
• Definition: Mosaicism – A condition where an individual has two or more genetically different sets of cells. In genome editing, this occurs if the edit happens after the first cell division, leaving some cells edited and others mutated.
• Key Finding: The embryos used the healthy maternal gene as a template for repair via the Homology-Directed Repair (HDR) pathway.

Question 17

Case study 3: First Gene Edited Babies

Answer 17

• C-C chemokine receptor type 5 (CCR5) , chemokine
• receptor involved in immune response.
• CCR5 is the main HIV coreceptor, involved in viral entry and cell-to-cell spread.
• CCR5 genetic polymorphisms associated with HIV-resistance.
• In 2018 Dr He Jiankui (associate professor of biophysics at SUSTech (China) claimed he had created the first genome edited CCR5 mutated twins (HIV father)
• largely condemned by the research community: conducted without scientific discussion, ethics, could have caused other genomic changes, CCR5 is required to fight other infections.
• Fired from SUSTech, three years in prison for “illegal medical practice”, 3 million yuan
• (£310,000)

Question 18

Pt 2

Answer 18

• In 2018, Dr. He Jiankui claimed to have created the first genome-edited human infants (twins).
• Target: The CCR5 gene, which encodes a chemokine receptor that serves as the main co-receptor for HIV entry into cells.
• The Goal: To create a mutation that mimics the natural $CCR5\Delta32$ polymorphism, which confers resistance to HIV.
• Ethical Condemnation: The research was globally condemned for several reasons:
• Conducted without proper scientific discussion or transparency.
• Lack of medical necessity (there are other ways to prevent HIV transmission).
• Risk of Off-target effects (unintended mutations in other parts of the genome).
• Creating “germline” changes that will be passed down to all future generations.

Question 19

Core Concepts in Clinical Editing

Answer 19

• Ex Vivo Editing: Cells (like blood stem cells) are removed from the patient, edited in a lab, and then returned to the patient.
• In Vivo Editing: The editing machinery (e.g., CRISPR carried by a virus) is injected directly into the patient’s body (e.g., the muscle or eye).
• Definition: Germline Editing – Editing the DNA of eggs, sperm, or embryos. These changes are heritable, meaning they are passed on to offspring.
• Definition: Somatic Editing – Editing the DNA of non-reproductive cells (e.g., muscle or liver). These changes are NOT heritable.

Question 20

Core Ethical Considerations

Answer 20

The rapid advancement of CRISPR/Cas9 and other editing tools has outpaced the development of international ethical frameworks. Key areas of concern include:
* Risk vs. Benefit Balance: Assessing whether the potential therapeutic benefits (e.g., curing a genetic disease) outweigh the risks of off-target mutations or long-term biological consequences.
* Liability: Determining who is responsible—scientists, clinicians, or manufacturers—if an edit goes wrong or causes unintended harm to a patient or their offspring.
* Ecological Disequilibrium: The concern that using genome editing in the wild (such as “gene drives” to eliminate malaria-carrying mosquitoes) could permanently alter or collapse entire ecosystems.

Question 21

Human Germline vs. Somatic Editing

Answer 21

A major ethical “red line” in the scientific community is the distinction between editing non-reproductive cells and reproductive cells.
* Definition: Somatic Editing – Changes made to the DNA of non-reproductive cells (e.g., blood, muscle, or liver cells). These changes affect only the individual patient and are not inheritable.
* Definition: Germline Editing – Changes made to the DNA of eggs, sperm, or early-stage embryos. These changes are inheritable, meaning they will be passed down to all future generations, raising concerns about “consent” for future individuals.

Question 22

Social Justice and Enhancement

Answer 22

Genome editing introduces the possibility of moving beyond “therapy” (curing disease) into “enhancement” (improving traits).
* The Social Divide: There is a significant concern that genome editing for enhancement (e.g., increased intelligence, physical strength, or longevity) will only be accessible to the wealthy.
* Consequence: This could create a permanent, biological “social divide” or a “genetic underclass,” exacerbating existing global inequalities.

Question 23

Specialized Applications

Answer 23

• Animal Chimeras: The creation of animals containing human cells or organs (often intended for organ transplantation/xenotransplantation). This raises questions regarding the “moral status” of these animals.
• Consumer Regulation: How to regulate genome-edited products in the food chain or direct-to-consumer genetic services.