explain that gene editing is a form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome

Principles of Genetic Technology (Cambridge AS & A Level Biology – Topic 19)

1. Scope of Genetic Technology – full list required by the syllabus

The syllabus expects students to be familiar with the following laboratory techniques. They are presented in the same order as the specification to aid quick cross‑referencing.

• Recombinant DNA technology – restriction‑enzyme digestion, ligation, plasmid vectors.
• Bacterial transformation – heat‑shock or electroporation of competent cells.
• Agrobacterium‑mediated plant transformation – Ti‑plasmid‑based gene transfer.
• Cloning – somatic‑cell nuclear transfer (SCNT) and embryo splitting.
• Gene‑transfer methods

  • Viral vectors (retroviral, lentiviral, adenoviral).
  • Particle bombardment (biolistics).
  • Microinjection (direct injection of DNA/RNA into cells or embryos).

• RNA interference (RNA‑i) and antisense technology – siRNA, hairpin RNA constructs, antisense oligonucleotides.
• Gene therapy – somatic‑cell and germ‑line approaches.
• Genome‑editing tools – zinc‑finger nucleases (ZFNs), transcription‑activator‑like effector nucleases (TALENs), CRISPR‑Cas systems, base editors, prime editors.
• Ethical, safety and regulatory issues – off‑target effects, germ‑line editing, ecological impact, intellectual‑property, and national/international legislation.

2. Molecular Basis of Gene Editing

2.1 Target‑site requirements (illustrated with CRISPR‑Cas9)

• PAM sequence: short motif required for Cas9 binding (NGG for S. pyogenes Cas9) located immediately downstream of the target.
• Guide RNA (gRNA) design: 20‑nt spacer complementary to the target; optimal GC content 40‑60 %; avoid mismatches to minimise off‑target cleavage.
• Off‑target prediction: use web‑based tools (e.g., CRISPOR, Benchling) to rank potential unintended sites and select the most specific gRNA.

2.2 Vector architecture for gene‑editing constructs

3. Gene‑Editing Workflow

1. Design the editing construct – choose target locus, design gRNA (or protein‑DNA binding domain for ZFNs/TALENs), assemble vector with nuclease, promoter, terminator, selectable marker, and donor template if HDR is required.
2. Delivery into cells – plant cells (Agrobacterium infection, particle bombardment), animal cells (electroporation, lipofection, microinjection, viral transduction).
3. Induction of a double‑strand break (DSB) – Cas9 (or other nuclease) creates a site‑specific cut; base/prime editors act without a DSB.
4. Cellular repair

   • Non‑homologous end joining (NHEJ) – error‑prone, generates indels → gene knock‑out.
   • Homology‑directed repair (HDR) – uses supplied donor → precise insertion or replacement.
   • Base editing – single‑base conversion without DSB.
   • Prime editing – small insertions, deletions or any base change without DSB.

5. Screening and validation – PCR, restriction‑fragment length analysis, Sanger or NGS sequencing, and/or phenotypic assays (e.g., fluorescence of a reporter).

4. DNA Repair Pathways Exploited in Editing

5. Comparison of Common Gene‑Editing Tools

6. Example Application – Correcting the Sickle‑Cell Mutation in the β‑Globin Gene (HBB)

1. Identify the mutation – A→T transversion in codon 6 (GAG → GTG).
2. Design a sgRNA that binds 20 nt upstream of an NGG PAM overlapping the mutant codon.
3. Prepare a donor template – single‑stranded DNA oligo containing the correct “A” and ~60 bp homology arms on each side.
4. Introduce editing components into patient‑derived haematopoietic stem cells by electroporation of a ribonucleoprotein (Cas9 protein + sgRNA) together with the donor oligo.
5. DSB formation and HDR – Cas9 cleaves the mutant allele; during S/G2 the cell uses the donor to repair, swapping the T for an A.
6. Selection and verification – PCR amplification of the target region followed by Sanger sequencing; optional use of a silent restriction site introduced in the donor for rapid screening.
7. Functional outcome – restored β‑globin synthesis; sickle‑cell phenotype abolished in differentiated erythrocytes in vitro.

7. Advantages of Gene Editing over Conventional Genetic Engineering

• Target specificity – edits are made at a defined locus, avoiding random insertion effects.
• Precision – single‑nucleotide changes are possible without adding foreign DNA.
• Speed and versatility – the same basic system can generate knock‑outs, knock‑ins, base changes or large insertions across many species.
• Regulatory benefit – when no foreign DNA remains (e.g., base‑edited crops), some jurisdictions treat the product similarly to conventionally bred varieties.

8. Ethical, Safety and Regulatory Considerations

• Off‑target mutations – must be assessed by whole‑genome sequencing or deep‑sequencing of predicted sites.
• Germ‑line editing – heritable changes; many countries prohibit clinical use until safety and societal consensus are achieved.
• Ecological impact – release of edited organisms (including gene‑drive constructs) requires thorough risk assessment.
• Intellectual‑property – patents on CRISPR‑Cas and related tools influence research collaboration and commercial exploitation.
• Regulatory frameworks – the UK, EU, USA and other regions have distinct classification schemes for GMOs versus gene‑edited organisms; students should be aware of the main differences.

9. Summary

Gene editing is a sophisticated branch of genetic technology that enables the insertion, deletion or replacement of DNA at precise genomic locations. By exploiting cellular repair pathways—NHEJ for knock‑outs, HDR for precise knock‑ins, and base/prime editing for single‑base changes—researchers can achieve targeted modifications with unprecedented accuracy. Mastery of the molecular requirements (PAM, guide design, full vector architecture) and an awareness of ethical, safety and regulatory issues are essential for success in both the laboratory and the Cambridge AS & A Level examinations.