Recombinant DNA, CRISPR, whole genome sequencing and gene therapy: HSC Biology Module 6
“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”
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.
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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.
- 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).
- The Cas9 nuclease binds the gRNA. The complex scans the genome and binds the matching target.
- Cas9 cuts both DNA strands at the target, producing a double-strand break.
- 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
| Technology | Year | What it does | Named example |
|---|---|---|---|
| Recombinant DNA | 1973 | Joins DNA from different sources | Humulin |
| Whole genome sequencing | 2003 first human | Reads all bases of a genome | Mendeliome diagnosis |
| Reproductive cloning | 1996 | Produces a genetic copy via SCNT | Dolly the sheep |
| Gene therapy | 1990 first trial | Inserts a working gene into a patient | Luxturna, Zolgensma |
| CRISPR-Cas9 | 2012 | Edits the genome at a precise location | Casgevy (sickle cell) |
Recombinant DNA inserts a gene; CRISPR edits a gene. Recombinant DNA technology uses restriction enzymes to cut a gene and a plasmid (leaving complementary sticky ends), DNA ligase to join them, and transformation to put the recombinant plasmid into a host that clones it (e.g. E. coli making insulin). CRISPR-Cas9 instead edits DNA in place: a guide RNA directs Cas9 to cut a precise target next to a PAM, and the cell's repair (error-prone NHEJ to knock out, or template-guided HDR to edit precisely) sets the outcome.
Worked example 1 - describe recombinant insulin production (4 marks)
Question. Describe how recombinant DNA technology is used to make human insulin in bacteria.
Model answer. A restriction enzyme cuts the human insulin gene from human DNA, leaving sticky ends. The same restriction enzyme cuts open a bacterial plasmid so its ends are complementary; the insulin gene is inserted and DNA ligase seals it in, forming a recombinant plasmid. The plasmid is taken up by a host bacterium (E. coli) by transformation. The bacteria reproduce, cloning the gene, and in fermenters they transcribe and translate it to make human insulin, which is purified for patients with diabetes.
Marker's note: 1 mark each for cutting the gene, ligating into the plasmid with the matched enzyme, transformation into a host, and expression/harvest. Naming tools without the cause/effect sequence caps below full marks.
Worked example 2 - explain the CRISPR mechanism (5 marks)
Question. Describe the CRISPR-Cas9 mechanism and explain how the repair pathway determines the outcome.
Model answer. A guide RNA complementary to a 20-base target (next to a PAM) loads into Cas9. The complex scans the genome, the gRNA base-pairs with the target, and Cas9 cuts both strands to make a double-strand break. If the cell repairs by non-homologous end joining (NHEJ), small indels are introduced that usually knock the gene out. If a donor template is supplied, homology-directed repair (HDR) copies it into the break, giving a precise edit or gene replacement. So the same cut either disables or corrects the gene, depending on the repair pathway.
Marker's note: 1 mark each for targeting (gRNA/PAM), the double-strand cut, NHEJ outcome, HDR outcome, and linking pathway to result. Band 6 answers contrast NHEJ and HDR explicitly.
Worked example 3 - compare two options for sickle cell disease (4 marks)
Question. A patient has sickle cell disease. Compare two genetic-technology options for treatment.
Model answer. Option 1 is recombinant gene therapy: a working beta-globin gene is delivered to the patient's haematopoietic stem cells using a lentiviral vector, and the returned cells produce functional haemoglobin (the Zynteglo approach). Option 2 is CRISPR editing: Cas9 with a guide RNA disrupts the BCL11A regulator in the patient's stem cells, reactivating foetal haemoglobin (which does not sickle), and the edited cells are returned (the Casgevy approach, approved 2023). Both can be curative; recombinant gene therapy adds a new gene that may integrate randomly, whereas CRISPR edits in place and avoids inserting a foreign gene, so it has fewer integration risks but carries an off-target editing risk.
Marker's note: marks for correctly naming and describing each technology, identifying a genuine point of comparison (insert versus edit; integration versus off-target risk), and tying each to its real example.
- 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.
- Name the tools precisely
- Restriction enzyme (cut), ligase (join), plasmid/vector (carry), transformation (uptake), guide RNA (target), Cas9 (cut), PAM (licence to bind). Vague terms like "genetic stuff" or "the machine" lose marks; precise terminology lifts a Band 4 answer to Band 6.
- Match the command word
- "Describe" wants the steps in order; "compare" wants the same features for both technologies (e.g. insert versus edit); "evaluate"/"assess" want a justified judgement weighing benefits against risks, anchored to a named example.
- Always attach a named example
- Insulin/Humulin (recombinant DNA), Casgevy (CRISPR), Luxturna/Zolgensma (gene therapy), Dolly/AquAdvantage salmon (cloning/transgenic). A named, correctly matched example is usually worth a mark.
- Keep recombinant DNA and CRISPR separate
- If a question says "edit at a precise locus", that is CRISPR; if it says "insert a gene into a vector and clone it", that is recombinant DNA.
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.
Think of a cell's DNA as a huge recipe book. Recombinant DNA technology is like photocopying one recipe (a gene), taping it into a tiny ring-binder (a plasmid), and slipping that binder into a bacterium so the bacterium keeps making millions of copies and cooks the dish for you - that is how we get insulin. CRISPR-Cas9 is different: instead of adding a page, it is a "find and replace" tool. A little RNA "search term" guides a protein called Cas9 to the exact line in the book, Cas9 snips it, and the cell either patches the snip messily (switching the gene off) or copies in a corrected line you supply (fixing it). Whole genome sequencing just reads the whole book to spot a typo, and gene therapy is the medicine that delivers a working page into a sick person's cells. Together these tools let us read, copy, edit and deliver genes - which is powerful, but also why people argue about where to draw the line.
Exam-style questions
Questions in the style of NESA exam questions on this dot point, each with a worked answer. They are written by ExamExplained unless tagged "Past paper"; the year shows the paper a question is modelled on.
2025 HSC-style4 marksAnopheles mosquitoes have been genetically modified to express PAI-1 (encoded by the human SERPINE 1 gene), which blocks malarial Plasmodium entering the mosquito gut. Describe a process that could be used to produce mosquitoes which express PAI-1.Show worked answer →
Describe recombinant DNA / transgenesis using correct named steps and tools. Sample answer: Use recombinant DNA technology to transfer the SERPINE 1 gene from a human to the mosquito. Restriction enzymes cut the SERPINE 1 gene from a human cell chromosome. A bacterial plasmid (circular DNA) is opened with the same restriction enzymes, and the SERPINE 1 gene is inserted and joined using DNA ligase. The bacteria reproduce, producing many copies of the gene (gene cloning). The gene is then delivered into the mosquito by micro-injection into mosquito egg cells, so the mosquitoes that develop contain SERPINE 1 and express PAI-1. Marks: 4 = describes an appropriate process; 3 = outlines an appropriate process; 2 = some understanding of how GM mosquitoes are produced; 1 = relevant information. Common error: describing breeding/cloning instead of transgenesis, and vague non-scientific terms instead of micro-injection/transformation.
Source: NESA 2025 HSC Biology examination and marking guidelines.
2023 HSC-style4 marksDescribe a named genetic technology and its use in a medical application.Show worked answer →
Name a technology, describe its steps with correct terminology, and match it to a medical use. Sample answer: Human insulin is produced by recombinant DNA technology to help diabetics. Restriction enzymes cut the insulin gene from a human cell. The same restriction enzyme is used to cut a section from a plasmid of E. coli so that the sticky ends are complementary. The plasmid is resealed with the insulin gene inserted (using DNA ligase), and the recombinant plasmid is inserted into a host to produce human insulin. The insulin is then used by patients to manage diabetes. Marks: 4 = comprehensive description of a named genetic technology AND its medical application; 3 = sound description; 2 = identifies and outlines a technology and application; 1 = relevant information. Common error: lacking specific terminology and an appropriately matched medical application.
Source: NESA 2023 HSC Biology examination and marking guidelines.
2021 HSC-style3 marksGenetically engineered Atlantic salmon carry a transgene combining a Chinook salmon growth-hormone coding sequence and an Ocean Pout antifreeze-protein promoter. Steps 1-4 of a diagram show the transgene being inserted into a plasmid and bacteria. Explain the processes shown in steps 1-4 (gene cloning using a plasmid and bacteria).Show worked answer →
Explain gene cloning with cause and effect and a clear purpose. Sample answer: The transgene is inserted into a plasmid using enzymes (restriction enzymes to cut and ligase to join). The plasmid is then placed into a bacterial host. As the host reproduces, the plasmid is copied and so is the transgene - this is gene cloning. This is done in order to produce multiple copies of the gene. Marks: 3 = explains the processes in the steps (cause/effect); 2 = outlines the process of gene cloning; 1 = relevant information. Common error: not using 'explain' (cause/effect), and failing to identify the purpose of gene cloning or the role of the plasmid and bacteria.
Source: NESA 2021 HSC Biology examination and marking guidelines.
Practice questions
Original practice questions graded from foundation to exam level, each with a full worked solution. Try them before revealing the solution.
foundation2 marksName the two enzymes used to build a recombinant plasmid and state the role of each.Show worked solution →
1 mark - restriction enzyme. A restriction enzyme (endonuclease) cuts DNA at a specific recognition sequence, releasing the gene of interest and opening the plasmid, often leaving complementary sticky ends.
1 mark - DNA ligase. DNA ligase forms phosphodiester bonds that seal the gene into the plasmid, producing a single recombinant DNA molecule.
Each enzyme must be tied to its job (cut versus join). Naming both enzymes without their functions caps at 1 mark.
foundation3 marksOutline the role of each of the following in CRISPR-Cas9 gene editing: (a) the guide RNA, (b) the Cas9 protein, (c) the PAM sequence.Show worked solution →
- 1 mark - guide RNA
- The guide RNA (gRNA) carries a ~20-base sequence complementary to the target DNA; it base-pairs with the target and so directs Cas9 to the correct site.
- 1 mark - Cas9
- Cas9 is a nuclease that cuts both strands of the DNA at the targeted site, producing a double-strand break.
- 1 mark - PAM
- The protospacer adjacent motif (PAM, usually NGG) is a short sequence immediately next to the target that Cas9 must recognise before it will bind and cut.
Each component needs its specific function (direct, cut, licence binding). Listing the parts without functions does not earn the marks.
foundation2 marksDistinguish between somatic and germline gene therapy.Show worked solution →
1 mark - somatic. Somatic gene therapy alters body (non-reproductive) cells, so the change is not inherited by the patient's offspring.
1 mark - germline. Germline gene therapy alters gametes or embryos, so the change is heritable and is banned in most jurisdictions on safety and consent grounds.
The discriminator is heritability (body cells versus gametes/embryos). Blurring the two caps at 1 mark.
core4 marksDescribe how recombinant DNA technology is used to produce human insulin in bacteria.Show worked solution →
- 1 mark - isolate the gene
- A restriction enzyme cuts the human insulin gene from human DNA, leaving complementary sticky ends.
- 1 mark - open and insert into a vector
- The same restriction enzyme cuts a bacterial plasmid, so its sticky ends match; the insulin gene is inserted and joined with DNA ligase, forming a recombinant plasmid.
- 1 mark - transformation
- The recombinant plasmid is taken up by a host bacterium (transformation, e.g. E. coli); the bacterium reproduces, copying the gene (gene cloning).
- 1 mark - expression and harvest
- Grown in fermenters, the transformed bacteria transcribe and translate the gene to make human insulin, which is purified for clinical use.
Full marks need the named tools (restriction enzyme, ligase, plasmid, host) AND the cause/effect sequence to the product. Omitting transformation or the purpose caps below full marks.
core4 marksCompare recombinant DNA technology and CRISPR-Cas9 as techniques for modifying an organism's genome.Show worked solution →
Award up to 4 marks for a genuine comparison (similarities AND differences), not two separate descriptions.
What each does (2 marks). Recombinant DNA technology inserts a new sequence (the gene of interest) into a vector such as a plasmid using restriction enzymes and ligase, and the insert lands at a non-specific / random site in the host genome. CRISPR-Cas9 edits DNA in place at a precise, chosen locus, using a guide RNA to direct Cas9 to cut, after which the cell's repair adds, removes or replaces bases.
Similarity and trade-off (2 marks). Both change the genotype and can give a new phenotype, and both can use vectors for delivery. However, CRISPR is more precise (targets one locus, can avoid leaving a bacterial backbone) whereas recombinant DNA reliably adds a whole functional gene that is then cloned in bulk. A relevant limitation (e.g. CRISPR off-target cuts) strengthens the comparison.
A response that describes each technique separately without explicit points of comparison caps below full marks.
core5 marksDescribe the mechanism of CRISPR-Cas9 gene editing, and explain how the choice of repair pathway determines the outcome.Show worked solution →
- 1 mark - targeting
- A guide RNA is designed to be complementary to a ~20-base target sequence that lies next to a PAM; the gRNA loads into Cas9.
- 1 mark - binding and cutting
- The Cas9-gRNA complex scans the genome, the gRNA base-pairs with the matching target, and Cas9 cuts both DNA strands, creating a double-strand break.
- 1 mark - NHEJ
- If the cell repairs the break by non-homologous end joining (NHEJ), the repair is error-prone and introduces small insertions or deletions (indels) that usually knock the gene out (disruption).
- 1 mark - HDR
- If a DNA repair template is supplied, the cell can use homology-directed repair (HDR) to copy the template into the break, giving a precise edit or gene replacement.
- 1 mark - link
- Therefore the same cut can either disable a gene (NHEJ) or correct/insert a sequence (HDR) depending on the repair pathway available.
Band 6 answers explicitly contrast the two repair pathways and link each to its outcome (knockout versus precise edit).
exam7 marksEvaluate the use of CRISPR-Cas9 gene editing in medicine, with reference to a named application.Show worked solution →
"Evaluate" requires a supported judgement weighing benefits against risks and limitations, anchored to a real example. A Band 6 answer reaches a reasoned conclusion, not just a list.
- The technology and a named application (2 marks)
- CRISPR-Cas9 uses a guide RNA to direct Cas9 to a precise locus, where it cuts and the cell's repair edits the gene. Casgevy (exa-cel), approved in 2023, treats sickle cell disease and beta-thalassaemia by editing a patient's own haematopoietic stem cells to knock out the BCL11A regulator, reactivating foetal haemoglobin (which does not sickle); the edited cells are reinfused.
- Benefits (2 marks)
- CRISPR is precise, can target the underlying cause rather than just symptoms, edits the patient's own cells (no donor/immune-match problem), and in trials over 90 percent of Casgevy patients stayed free of pain crises - a potentially curative, one-time treatment.
- Risks and limitations (2 marks)
- Off-target edits at similar sequences, mosaicism, the requirement for chemotherapy/bone-marrow ablation, very high cost (millions of dollars per patient, raising equity-of-access concerns), and the ethical line that germline editing must not be crossed.
- Judgement (1 mark)
- A justified conclusion - e.g. for serious monogenic diseases with no good alternative, the benefits outweigh the risks for somatic therapy under strict regulation and off-target screening, while germline use remains unjustified. Listing pros and cons without an explicit judgement caps below full marks.
exam6 marksGenetically modified crops and cloned transgenic livestock are increasingly used in agriculture. Assess the benefits and risks of producing transgenic species using genetic technologies.Show worked solution →
"Assess" needs a weighed judgement of benefits against risks across named examples.
- How transgenic species are made (1-2 marks)
- A gene of interest is inserted by recombinant DNA technology (restriction enzymes, ligase, a vector such as Agrobacterium for plants or micro-injection for animals) or edited by CRISPR; somatic cell nuclear transfer (SCNT) can then clone a successful transgenic animal to copy the genotype reliably (e.g. Dolly-style cloning, GM AquAdvantage salmon, GloFish).
- Benefits (1-2 marks)
- Improved yield and traits (faster-growing salmon, pest- or mildew-resistance, polled cattle avoiding dehorning), pharmaceuticals in milk, and uniform stock from cloning.
- Risks (1-2 marks)
- Reduced genetic diversity in cloned/uniform populations (vulnerable to disease), ecological risk if GM organisms escape and breed with wild populations, low success rates and developmental abnormalities in mammalian cloning, and ethical/welfare and consumer-acceptance concerns.
- Judgement (1 mark)
- A supported conclusion weighing food-security and welfare benefits against biodiversity and ecological risk, typically concluding such technologies are justified under regulation and containment but require ongoing monitoring. A list without a judgement caps below full marks.
Practise this
Sources & how we know this
- Biology Stage 6 Syllabus (2017) — NESA
- Biology HSC exam papers — NESA