Biology • Year 12 • Module 6 • Lesson 13
Current Genetic Technologies That Induce Genetic Change
Apply the lesson's three-category framework (reproductive, DNA-copying, DNA-editing) to real comparison data from three genome-editing technologies: ZFNs, TALENs and CRISPR-Cas9.
1. Compare three DNA-editing technologies — real cleavage-efficiency data
The table below compares three site-specific DNA-editing technologies: zinc-finger nucleases (ZFNs), TALENs and CRISPR-Cas9. All three are DNA-level technologies in the lesson's framework — each makes a targeted double-strand break that the cell then repairs, producing an edit at the chosen site. The cleavage / cut-rate figures below are typical of the values reported in peer-reviewed studies in mammalian cells. 7 marks
| Technology | Year introduced | Targeting mechanism | Typical cleavage / edit rate at target site (%) | Off-target activity | Approximate reagent design time |
|---|---|---|---|---|---|
| ZFNs | 2003 | Protein–DNA recognition (each finger = 3 bp) | ~ 10 % | Moderate | Months |
| TALENs | 2011 | Protein–DNA recognition (one TALE repeat = 1 bp) | ~ 20 % | Lower than ZFNs | Weeks |
| CRISPR-Cas9 | 2012 | RNA–DNA base-pairing (a 20-nt guide RNA) | ~ 40–80 % | Variable; can be high without engineering | Days |
Data adapted from Gaj, Gersbach & Barbas (2013) Trends in Biotechnology 31(7): 397–405, and Ran et al. (2013) Nature Protocols 8: 2281–2308. Figures are typical values reported in mammalian cells.
1.1 Identify the technology with the highest typical cleavage rate at the target site, and quote a supporting figure from the table. 2 marks
1.2 Using the table, describe two reasons why CRISPR-Cas9 spread through laboratories far faster than ZFNs after 2012. Refer to specific values. 3 marks
1.3 All three technologies appear in the same lesson category — DNA-level genetic technologies. Explain one important way they all differ from artificial insemination, despite all being "genetic technologies". 2 marks
2. Interpret graph — efficiency vs design time
The graph below plots typical reported cleavage efficiency at the target site (%) against approximate reagent design time (days, log scale) for the three editing technologies in mammalian cells. 6 marks
Plot constructed from typical mammalian-cell values in Gaj et al. (2013) and Ran et al. (2013).
2.1 Describe the trend the dashed arrow summarises. Refer to both axes. 2 marks
2.2 A research team has only a fortnight to set up a knock-out of one gene in mouse cells. Using the graph, justify which technology you would recommend, and explain why the others are less suitable. 2 marks
2.3 The lesson warns against treating all genetic technologies as "the same thing". Use the graph to show why distinguishing within a category matters, not just between categories. 2 marks
3. Case studies — three real uses of genome-editing technologies
Read each short case study, then answer the question that follows. Use lesson terminology (reproductive technology · DNA copying · DNA insertion · induced genetic change). 9 marks
Case A — ZFNs and sickle-cell disease (Sangamo trials, 2014–2020).
A clinical trial used zinc-finger nucleases delivered to a patient's haematopoietic stem cells ex vivo to disrupt the BCL11A erythroid enhancer. Disrupting this enhancer re-activates fetal haemoglobin, partially compensating for the sickle-cell mutation. Cleavage at the target site was reported at approximately 30–40% of edited cells after optimisation.
3.1 In the lesson's framework, identify the category of genetic technology being used here, and explain what is being "induced" — at what biological level. 3 marks
Case B — TALENs in cattle (Carlson et al., 2016).
Researchers used TALENs to introduce the naturally-occurring POLLED allele into Holstein dairy cattle. The edit removed horn development without crossing to a different beef breed, preserving the dairy genetics. Edit efficiency at the target locus reached ~20% of treated cells; selected edited founders were used to establish a hornless dairy line by conventional breeding.
3.2 The researchers combined a DNA-level technology (TALENs) with a reproductive technology (artificial insemination of the founder line). Explain why both categories were required, using the table data from Section 1. 3 marks
Case C — CRISPR-Cas9 and Casgevy (FDA approval, 2023).
Casgevy (exagamglogene autotemcel) is a CRISPR-Cas9 therapy approved by the US FDA in December 2023 for sickle-cell disease and transfusion-dependent β-thalassaemia. It uses an ex vivo guide-RNA / Cas9 ribonucleoprotein to disrupt the same BCL11A enhancer that the earlier ZFN trial targeted. Reported on-target editing efficiency in patient HSCs is ~80%.
3.3 Casgevy and the ZFN approach in Case A target the same DNA sequence and produce the same clinical aim. Using both the table data and the lesson's "what changes, where it acts, why used" framework, justify why CRISPR-Cas9 — not ZFNs — was the technology that reached FDA approval first for this disease. 3 marks
4. Apply — sort six technologies into the three lesson categories
A Year 12 student is preparing notes for the IQ3 entry essay. Their list has six items, but they have not yet sorted them. Place each item in the correct row of the table below, and write a one-sentence justification using lesson terminology. 6 marks (1 sort + 1 justification per item)
Items to sort: CRISPR-Cas9 editing of BCL11A · embryo transfer in cattle · gene cloning into a plasmid · artificial pollination of orchids · production of a transgenic Bt cotton line · somatic-cell nuclear transfer (whole-organism cloning).
| Lesson category | Item | One-sentence justification |
|---|---|---|
| Reproductive technology (controls gamete combination) | ||
| Reproductive technology (controls gamete combination) | ||
| DNA-level — copies DNA | ||
| DNA-level — inserts or edits DNA | ||
| DNA-level — inserts or edits DNA | ||
| Cellular / developmental — preserves a genotype |
Q1.1 — Highest cleavage rate (2 marks)
CRISPR-Cas9 has the highest typical cleavage rate at the target site [1], reported at approximately 40–80% in mammalian cells, compared with ~20% for TALENs and ~10% for ZFNs [1].
Marking notes. 1 mark for naming CRISPR-Cas9; 1 mark for quoting a supporting figure from the table.
Q1.2 — Why CRISPR spread fastest (3 marks)
(i) Design time. CRISPR-Cas9 reagents can be designed in days because targeting uses a short guide RNA whose sequence is changed by ordering a new oligonucleotide, while ZFNs require months of protein engineering to retarget [1 — refer to "days" vs "months"]. (ii) Efficiency. CRISPR-Cas9 typically achieves ~40–80% cleavage compared with ~10% for ZFNs, so fewer cells need to be screened to recover a successful edit [1 — quote ~40–80% vs ~10%]. (iii) Combined effect. The two together mean a lab can attempt many targets per year using CRISPR for the same effort that one ZFN target used to require [1 — synthesises both factors].
Marking notes. 1 mark for each correct reason with a supporting figure; 1 mark for combining both into a "many more experiments per year" or equivalent synthesis. Max 3.
Q1.3 — How DNA-editing differs from artificial insemination (2 marks)
ZFNs, TALENs and CRISPR all change DNA sequence directly at a chosen site within the genome [1]. Artificial insemination is a reproductive technology — it only controls which sperm fertilises which egg; it does not insert, copy or alter a DNA sequence, so it cannot introduce a trait that is not already present somewhere in the species' allele pool [1].
Q2.1 — Trend (2 marks)
As reagent design time decreases (CRISPR ~5 days, TALENs ~14 days, ZFNs ~90 days), cleavage efficiency at the target site increases (~10% → ~20% → ~60%) [1]. The newer technologies are both faster to design and more efficient per attempt [1].
Q2.2 — Two-week deadline (2 marks)
Choose CRISPR-Cas9. Its design time of ~5 days easily fits inside a fortnight, while TALENs (~14 days) leaves no margin and ZFNs (~90 days) cannot be designed in time at all [1]. The higher cleavage efficiency (~60%) also means fewer cell-sorting rounds, so the editing step itself finishes faster — and the deadline is met [1].
Q2.3 — Within-category distinctions matter (2 marks)
All three technologies are DNA-editing tools, yet they differ in efficiency by an order of magnitude and in design time by a factor of about 20 [1]. So the choice of which DNA-editing technology to use has consequences for cost, speed and feasibility — students should not collapse "DNA editing" into one undifferentiated label any more than they should collapse "reproductive technology" with "DNA editing" [1].
Q3.1 — Sickle-cell ZFN trial (3 marks)
Category: DNA-level genetic technology — DNA editing / insertion [1]. What is induced: a double-strand break is made by the ZFN at a chosen sequence within the BCL11A enhancer in the patient's haematopoietic stem cells, and repair errors disrupt the enhancer [1]. Level of action: DNA sequence within somatic stem cells ex vivo — not at the level of reproduction and not aimed at producing a whole new organism [1].
Marking notes. 1 mark for correct category; 1 mark for identifying that a targeted DNA change is induced; 1 mark for identifying the level (somatic DNA, not germline / reproduction).
Q3.2 — Combining TALENs + AI in hornless dairy cattle (3 marks)
TALENs were needed because the POLLED allele had to be introduced into a Holstein dairy genome without crossing in a beef-breed background — only a DNA-level technology can do that directly [1]. However, TALEN edit efficiency in the table is ~20%, so even after editing only a fraction of founder cells carried the desired allele [1]. Artificial insemination is then used as a reproductive technology to multiply the successfully-edited founder across the dairy population, spreading the new allele rapidly without re-editing each animal [1].
Q3.3 — Why CRISPR (Casgevy) reached approval first (3 marks)
Both CRISPR and ZFNs target the same BCL11A enhancer in haematopoietic stem cells, but they differ on the three criteria the lesson uses to evaluate technologies: control, efficiency and targeted outcome [1]. CRISPR-Cas9 achieves ~80% editing in patient HSCs versus ~30–40% for the ZFN approach, so a much higher fraction of infused cells will produce fetal haemoglobin after engraftment — this is the "efficiency" axis [1]. CRISPR reagents can be designed in days using a guide RNA, so multiple targeting strategies could be screened quickly during preclinical development — this is the "control / iteration speed" axis. Together, higher efficiency + faster iteration made CRISPR the technology that crossed the regulatory bar first, in December 2023 [1].
Q4 — Sort six technologies (6 marks)
Reproductive technologies (1 mark each, max 2): embryo transfer in cattle — manipulates which embryo is gestated, without editing DNA; artificial pollination of orchids — controls which pollen reaches the stigma.
DNA-level — copies DNA (1 mark): gene cloning into a plasmid — copies a selected sequence using a vector and host cell.
DNA-level — inserts / edits DNA (1 mark each, max 2): CRISPR-Cas9 editing of BCL11A — targeted DNA change at a chosen site; production of a transgenic Bt cotton line — inserts a chosen Bt toxin gene from Bacillus thuringiensis.
Cellular / developmental — preserves a genotype (1 mark): somatic-cell nuclear transfer (whole-organism cloning) — preserves the donor's nuclear genotype without sexual reshuffling.
Marking notes. 1 mark per correct placement (max 6). Justifications must reference what each technology changes (gamete combination / DNA copy / DNA edit / genotype preservation).