Investigate the uses and applications of genetic technologies (past, present and future), including: recombinant DNA technology, CRISPR-Cas9, whole genome sequencing, gene therapy and cloning of transgenic species

What this dot point is asking

NESA wants you to know the named genetic technologies, their mechanisms and at least one application of each. The technologies overlap (CRISPR is often delivered using recombinant viral vectors), so be careful to identify what each technique uniquely does.

The answer

Recombinant DNA technology

The classical biotechnology toolkit, established in the 1970s. It combines DNA from different sources into a single molecule.

Key tools.

• Restriction enzymes (endonucleases). Cut DNA at specific palindromic sequences. EcoRI cuts at GAATTC and leaves single-stranded "sticky ends" that base-pair with complementary fragments.
• DNA ligase. Forms phosphodiester bonds that seal the cut DNA, joining the gene of interest into the vector.
• Plasmid vectors. Circular bacterial DNA carrying an origin of replication, the inserted gene, and a selectable marker (e.g. antibiotic resistance).
• Transformation hosts. E. coli for bacterial expression, yeast for eukaryotic post-translational modifications, Agrobacterium tumefaciens for plant cells.

Worked example. Recombinant human insulin: the human insulin gene is cut with restriction enzymes, ligated into a plasmid, transformed into E. coli, and grown in industrial fermenters; the bacteria secrete human insulin, which is purified for clinical use.

CRISPR-Cas9

Discovered as a bacterial immune system; reprogrammed for genome editing by Doudna and Charpentier (Nobel Prize 2020).

Mechanism.

1. A guide RNA (gRNA) is synthesised to match a 20-base target sequence in the genome, located next to a PAM (protospacer adjacent motif, usually NGG).
2. The Cas9 nuclease binds the gRNA. The complex scans the genome and binds the matching target.
3. Cas9 cuts both DNA strands at the target, producing a double-strand break.
4. The cell repairs the break:
   • Non-homologous end joining (NHEJ). Quick but error-prone; introduces small indels that often knock the gene out (gene disruption).
   • Homology-directed repair (HDR). A supplied DNA template is copied into the break, enabling precise gene editing or replacement.

Applications. Knock-out cell lines for research, agricultural traits (mildew-resistant wheat, polled cattle, mushroom browning), gene therapy (Casgevy, approved 2023 for sickle cell disease and beta-thalassaemia).

Whole genome sequencing (WGS)

What it is. Reading every base of an organism's genome, usually using next-generation sequencing technologies that read millions of short fragments in parallel and assemble them computationally.

Applications.

• Medical diagnosis of rare inherited disease (Mendeliome panels).
• Pharmacogenomics to guide drug choice (CYP variants).
• Cancer genomics identifying driver mutations and matching targeted therapies.
• Population genetics and ancestry.
• Pathogen surveillance during outbreaks (COVID-19, antimicrobial resistance tracking).
• Agriculture and conservation. Sequencing crop and livestock genomes for marker-assisted selection; sequencing endangered species to manage inbreeding.

Cost has fallen from US$3 billion for the first human genome (2003) to under US$200 today, enabling routine clinical use.

Gene therapy

What it is. Inserting, correcting or silencing a gene in a patient's cells to treat a genetic disease.

Delivery methods.

• Viral vectors. Adeno-associated virus (AAV) and lentivirus carry the therapeutic gene into cells.
• Lipid nanoparticles. Used for mRNA-based therapies and some CRISPR delivery.
• Ex vivo editing. Cells (often haematopoietic stem cells or T cells) are removed, edited in culture and returned to the patient.

Somatic vs germline.

• Somatic gene therapy alters body cells; the change is not passed to offspring. Widely accepted.
• Germline gene therapy alters gametes or embryos; the change is heritable. Banned in most jurisdictions because of consent and safety issues.

Worked examples.

• Luxturna. AAV delivery of RPE65 to retinal cells in patients with inherited retinal dystrophy.
• Zolgensma. AAV delivery of SMN1 for spinal muscular atrophy.
• CAR-T cells (Kymriah, Yescarta). A patient's T cells are removed, engineered to express a tumour-targeting receptor, and reinfused to attack leukaemia or lymphoma.

Cloning of transgenic species

Reproductive cloning. Somatic cell nuclear transfer (SCNT): the nucleus of a somatic cell is inserted into an enucleated egg, producing an embryo genetically identical to the donor.

Worked examples.

• Dolly the sheep (1996). First mammal cloned by SCNT.
• Transgenic dairy cattle (Daisy, 2012). Cloned cows expressing a human milk protein.
• GloFish. Zebrafish transgenic for jellyfish GFP, sold as ornamental fish; the first GM pet.

Reproductive cloning of mammals is technically demanding, with low success rates and developmental abnormalities.

Summary table

Worked example

You have a patient with sickle cell disease. Compare two genetic-technology options.

Option 1: Recombinant gene therapy. A working beta-globin gene is delivered to haematopoietic stem cells using a lentiviral vector. The cells are returned to the patient and produce functional haemoglobin. This is the approach used by Zynteglo.

Option 2: CRISPR editing. CRISPR-Cas9 is used to disrupt the BCL11A gene in the patient's stem cells, reactivating fetal haemoglobin (HbF), which is not affected by the sickle mutation. The edited cells are returned to the patient. This is the approach used by Casgevy (approved 2023).

Both work; Casgevy avoids inserting a new gene and so has fewer integration risks.

Common traps

Treating recombinant DNA and CRISPR as the same thing. Recombinant DNA inserts new sequence into a vector; CRISPR edits existing sequence in place.

Confusing somatic and germline therapy. Somatic = body cells, not heritable; germline = gametes or embryos, heritable and largely banned.

Saying CRISPR has no off-target effects. It has fewer than older editing tools, but off-target cuts at similar sequences are a real risk that researchers actively screen for.

Listing WGS as a treatment. WGS is a diagnostic technology; it tells you what is wrong, but does not directly treat anything.

In one sentence

Modern genetic technologies use restriction enzymes and ligase to build recombinant DNA in plasmid vectors, Cas9 and a guide RNA to edit precise sites with CRISPR, next-generation sequencers to read whole genomes for diagnosis and pharmacogenomics, and viral or lipid vectors to deliver therapeutic genes (or edited cells) in somatic gene therapy.