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

Examples in context

Example 1. Casgevy (exa-cel) for sickle cell disease in 2024. The first CRISPR-Cas9 therapy approved by the US FDA and UK MHRA, Casgevy treats sickle cell disease by editing the patient's own bone marrow stem cells. Clinicians extract haematopoietic stem cells from the patient, deliver Cas9 with a guide RNA targeting the BCL11A regulatory region, knock out that region to reactivate foetal haemoglobin (which does not sickle), and reinfuse the edited cells after chemotherapy ablates the patient's existing bone marrow. Over 90 percent of treated patients have remained free of pain crises in trials. Australia's TGA is currently evaluating Casgevy for PBS listing; if approved the per-patient cost is roughly 3.5 million AUD, raising equity-of-access questions.

Example 2. Whole genome sequencing of newborns in NSW pilot programs. Since 2023, the Sydney Children's Hospitals Network has piloted whole genome sequencing as part of newborn screening, alongside the traditional heel-prick metabolic test. Each baby's genome is sequenced for around 1000 AUD, generating roughly 100 GB of raw data per infant. Bioinformatics pipelines filter the 4 to 5 million variants in each genome against the ClinVar database to flag actionable variants in genes such as PAH (phenylketonuria), CFTR (cystic fibrosis) and SCN1A (Dravet syndrome). Babies with high-impact variants are referred for confirmatory testing and treatment, often before symptoms appear, dramatically improving outcomes for previously undetected disorders.

Try this

Q1. State the function of each of the following components in CRISPR-Cas9 gene editing: (a) the guide RNA, (b) the Cas9 protein, (c) the PAM sequence. [3 marks]

• Cue. (a) Directs Cas9 to the target DNA by base pairing. (b) Cuts both DNA strands. (c) Protospacer adjacent motif required for Cas9 binding immediately adjacent to the target.

Q2. A pharmaceutical company sequences 500 patient genomes for a new pharmacogenomic test. If each genome is 3.2 billion base pairs and average sequencing coverage is 30 times, calculate the total number of base pairs sequenced and explain why high coverage is needed. [3 marks]

• Cue. 500 by 3.2 billion by 30 = 48 trillion bp. High coverage reduces sequencing error and improves variant calling confidence at heterozygous positions.

Q3. Compare recombinant DNA technology with CRISPR-Cas9 for producing a transgenic organism. (a) Identify one mechanism difference. (b) Identify one advantage of CRISPR over restriction-enzyme cloning. (c) Identify one risk of CRISPR that is shared or unique. [1+2+2 marks]

• Cue. (a) Recombinant DNA inserts new sequences at random; CRISPR can edit at a precise locus. (b) Precision, multiplexing, no foreign DNA backbone. (c) Off-target editing, mosaicism, germline transmission.