IMAT Biology · Microorganisms and Biotechnology
GM Organisms, Gene Therapy, Stem Cells, Cloning and CRISPR
The tools of DNA technology put to use: transgenic microbes, crops and animals, adding working genes to patients, stem cells, clones and precise gene editing.
Why this matters
Two Cambridge papers asked about uses: which combinations of DNA a transgenic organism can carry (2017), and why gene therapy for cystic fibrosis gives only short-term relief (2018). Stem cells, cloning and CRISPR have not been asked yet in this chapter.
Concept 1 of 5: Genetically modified organisms and their products
A gene is a set of instructions written in a code that almost every organism reads the same way. So a gene taken from a bacterium, a jellyfish or a human can be read and turned into the same protein by a plant, a mouse or a yeast. That shared code is what makes transgenic organisms possible.
Definition
- A transgenic (genetically modified) organism contains DNA from another species. Any combination is possible: bacterial genes in plants, plant genes in animals, human genes in bacteria or goats.
- This works because the genetic code is universal: the same codons mean the same amino acids in nearly all organisms.
- Bacteria cannot remove introns, so eukaryotic genes go into bacteria as cDNA.
- Human insulin was the first medicine made this way (approved in 1982). Before that, diabetics used insulin from pigs and cattle.
- Concerns: genes spreading to wild relatives, pests becoming resistant to built-in toxins, possible allergens, and control of seeds by a few companies. The EU regulates GM crops strictly.
| Product or organism | Gene added | Host | Purpose |
|---|---|---|---|
| Human insulin | Human insulin gene (as cDNA) | E. coli or yeast | Treat type 1 diabetes |
| Bt cotton, Bt maize | Toxin gene from the bacterium Bacillus thuringiensis | Plant, using Agrobacterium | Kills insect larvae that eat the crop |
| Golden Rice | Genes for making β-carotene | Rice | Reduces vitamin A deficiency |
| Herbicide-tolerant soya | A bacterial gene the herbicide cannot block | Soya | Weeds can be sprayed without harming the crop |
| Hepatitis B vaccine | Gene for a viral surface protein | Yeast | A safe vaccine antigen |
| Transgenic goats | Human antithrombin gene, active in the udder | Goat | A human medicine collected from the milk |
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Transgenic DNA can cross any kingdom
There is no rule limiting gene transfer to related species. Invertebrate genes in mammals, bacterial genes in plants and plant genes in animals have all been made. Options that allow only one direction, or only prokaryote to eukaryote, are wrong.
Concept 2 of 5: Gene therapy: somatic and germline
Many genetic diseases happen because a person has two faulty recessive alleles and so no working protein. Adding one working copy of the gene to the cells that need it can be enough, because the working allele is dominant. The difficulty is getting the gene in, and keeping it there.
Definition
- Gene therapy adds a working allele to a patient's cells, using a vector: a modified virus or a liposome.
- Somatic gene therapy changes body cells only; it is not inherited. Germline gene therapy would change eggs, sperm or embryos, so it would be inherited; it is banned in most countries.
- It suits recessive single-gene disorders: one working allele restores the protein. The first approved trial (1990) treated SCID, a severe immune deficiency.
- Cystic fibrosis is caused by faulty alleles of the CFTR gene, which codes for a chloride channel. Working alleles can be delivered to airway cells by liposomes or viral vectors in an aerosol.
- Problems: cells that receive the gene die and are replaced by new cells from the patient's own stem cells, which still carry the faulty alleles, so treatment must be repeated; viral vectors can cause immune reactions; a gene inserted in the wrong place can disrupt another gene.
| Feature | Somatic gene therapy | Germline gene therapy |
|---|---|---|
| Cells changed | Body cells, such as airway or blood cells | Eggs, sperm or early embryo |
| Passed to children | No | Yes |
| How long it lasts | Only as long as the treated cells live | Every cell of the new person carries it |
| Status | Approved treatments exist | Banned in most countries |
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The added allele is dominant, and it fades with the cells
The working allele added in gene therapy is the dominant one, which is why one copy helps. The effect is short-lived because treated cells are replaced by new ones made from untreated stem cells, not because genes break down on their own in a cell.
Concept 3 of 5: Stem cells and potency
A stem cell has not yet decided what to become, and it can divide to make more of itself. The earlier in development a cell is, the more options it keeps open. A fertilised egg can make an entire person plus the placenta; a bone marrow stem cell can only make blood cells.
Definition
- A stem cell is unspecialised, can self-renew by dividing, and can differentiate into specialised cells.
- Potency is the range of cells it can become (see the table).
- Embryonic stem cells come from the inner cell mass of a blastocyst (about 5 days after fertilisation). Using embryos raises ethical objections.
- Induced pluripotent stem (iPS) cells are adult body cells reprogrammed to pluripotency by adding a few genes for transcription factors (Yamanaka, 2006). Made from the patient's own cells, they avoid rejection and the embryo question.
- Uses: bone marrow transplants (already routine), research into repairing damaged tissue.
- In plants, meristems hold stem cells, and many mature plant cells can become totipotent again in tissue culture.
| Potency | Can become | Examples |
|---|---|---|
| Totipotent | Every cell type, including the placenta | The zygote and the cells of the first few divisions |
| Pluripotent | Any cell of the body, but not the placenta | Embryonic stem cells from the inner cell mass; iPS cells |
| Multipotent | Several cell types of one family | Bone marrow stem cells, which make all blood cells |
| Unipotent | Only one cell type | Muscle satellite cells, which make muscle fibres |
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Pluripotent is one step below totipotent
Only the zygote and the cells of the first few divisions are totipotent: they can also form the placenta. Embryonic stem cells from the inner cell mass are pluripotent: any body cell, but not the placenta.
Concept 4 of 5: Cloning: natural, plant and animal
A clone is a genetically identical copy. Nature makes clones all the time, every time a bacterium divides or a strawberry plant sends out a runner. Cloning an adult mammal is harder: the nucleus of a specialised cell has to be put into an egg, which resets it to start development again.
Definition
- A clone is a group of genetically identical cells or organisms, made by asexual reproduction or mitosis.
- Natural clones: bacteria (binary fission), plants that spread by runners or tubers, and identical twins (one early embryo splits).
- Somatic cell nuclear transfer (SCNT): the nucleus of a body cell from the animal to be copied is put into an egg cell whose own nucleus has been removed. An electric pulse fuses them and starts division; the embryo is placed in a surrogate mother.
- Dolly the sheep (born 1996) was the first mammal cloned from an adult cell: the nucleus came from an udder cell.
- The clone shares the nuclear DNA of the nucleus donor, but its mitochondrial DNA comes from the egg donor. The surrogate gives no DNA.
- Therapeutic cloning grows an SCNT embryo only to the blastocyst stage to obtain embryonic stem cells matched to a patient.
| Type of cloning | How it is done | Example |
|---|---|---|
| Natural cloning | Asexual reproduction, or splitting of an early embryo | Bacteria, strawberry runners, identical twins |
| Plant cloning | Cuttings, or tissue culture of small pieces of tissue | Mass production of identical crop plants |
| Reproductive cloning (SCNT) | Body-cell nucleus into an enucleated egg; embryo into a surrogate | Dolly the sheep, 1996 |
| Therapeutic cloning | SCNT embryo grown only to a blastocyst | Embryonic stem cells matched to a patient |
| Gene cloning | Many copies of one gene made in bacteria or by PCR | Bacteria carrying a recombinant plasmid |
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A clone is not a perfect copy of the nucleus donor
A cloned animal has the donor's nuclear DNA but the egg donor's mitochondrial DNA, and it grows up in a different environment. So it can differ from the donor in small ways, including its appearance and behaviour. The surrogate mother gives it no DNA at all.
Concept 5 of 5: CRISPR-Cas9 gene editing in outline
CRISPR is a cut-and-repair tool that can be aimed at almost any gene. A short guide RNA does the aiming by base pairing with the target DNA, and the Cas9 enzyme it carries does the cutting. The cell then repairs the cut, and the way it repairs it decides whether a gene is switched off or rewritten.
Definition
- CRISPR began as a bacterial defence system: bacteria store short pieces of phage DNA and use matching RNAs to guide Cas enzymes to cut the phage if it returns.
- Cas9 is a nuclease that cuts both strands of DNA. It goes where the guide RNA (about 20 nucleotides long) pairs with the DNA, next to a short PAM sequence.
- Repair by simply joining the ends often adds or loses a few bases, which knocks out the gene. Repair copying a supplied DNA template makes a precise edit or inserts new DNA.
- Nobel Prize in Chemistry 2020: Emmanuelle Charpentier and Jennifer Doudna.
- The first approved CRISPR treatment (2023) edits a patient's own blood stem cells outside the body to treat sickle cell disease and β-thalassaemia.
- Editing embryos (germline editing) is widely condemned; a 2018 attempt in China led to a prison sentence.
| Component | What it is | Job |
|---|---|---|
| Cas9 | A nuclease enzyme (protein) | Cuts both strands of the target DNA |
| Guide RNA | About 20 nucleotides matching the target | Leads Cas9 to the right place by base pairing |
| PAM | A short DNA sequence beside the target | Must be present for Cas9 to cut |
| End-joining repair | The cell rejoins the cut ends, often imperfectly | Small insertions or deletions knock the gene out |
| Template repair | The cell copies a DNA template supplied with Cas9 | A precise change or an inserted sequence |
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The guide RNA aims; Cas9 cuts
Apart from needing a short PAM beside the target, Cas9 has no fixed target sequence, unlike a restriction enzyme. The guide RNA chooses the site by base pairing, so one Cas9 can be sent to almost any gene just by changing the guide.
Summary — formulas & gotchas at a glance
A revision cheat-sheet for the formulas and gotchas above. Click any concept name to jump back to its full explanation.
Reference tables (5)
Genetically modified organisms and their products6 rows
| Product or organism | Gene added | Host | Purpose |
|---|---|---|---|
| Human insulin | Human insulin gene (as cDNA) | E. coli or yeast | Treat type 1 diabetes |
| Bt cotton, Bt maize | Toxin gene from the bacterium Bacillus thuringiensis | Plant, using Agrobacterium | Kills insect larvae that eat the crop |
| Golden Rice | Genes for making β-carotene | Rice | Reduces vitamin A deficiency |
| Herbicide-tolerant soya | A bacterial gene the herbicide cannot block | Soya | Weeds can be sprayed without harming the crop |
| Hepatitis B vaccine | Gene for a viral surface protein | Yeast | A safe vaccine antigen |
| Transgenic goats | Human antithrombin gene, active in the udder | Goat | A human medicine collected from the milk |
Gene therapy: somatic and germline4 rows
| Feature | Somatic gene therapy | Germline gene therapy |
|---|---|---|
| Cells changed | Body cells, such as airway or blood cells | Eggs, sperm or early embryo |
| Passed to children | No | Yes |
| How long it lasts | Only as long as the treated cells live | Every cell of the new person carries it |
| Status | Approved treatments exist | Banned in most countries |
Stem cells and potency4 rows
| Potency | Can become | Examples |
|---|---|---|
| Totipotent | Every cell type, including the placenta | The zygote and the cells of the first few divisions |
| Pluripotent | Any cell of the body, but not the placenta | Embryonic stem cells from the inner cell mass; iPS cells |
| Multipotent | Several cell types of one family | Bone marrow stem cells, which make all blood cells |
| Unipotent | Only one cell type | Muscle satellite cells, which make muscle fibres |
Cloning: natural, plant and animal5 rows
| Type of cloning | How it is done | Example |
|---|---|---|
| Natural cloning | Asexual reproduction, or splitting of an early embryo | Bacteria, strawberry runners, identical twins |
| Plant cloning | Cuttings, or tissue culture of small pieces of tissue | Mass production of identical crop plants |
| Reproductive cloning (SCNT) | Body-cell nucleus into an enucleated egg; embryo into a surrogate | Dolly the sheep, 1996 |
| Therapeutic cloning | SCNT embryo grown only to a blastocyst | Embryonic stem cells matched to a patient |
| Gene cloning | Many copies of one gene made in bacteria or by PCR | Bacteria carrying a recombinant plasmid |
CRISPR-Cas9 gene editing in outline5 rows
| Component | What it is | Job |
|---|---|---|
| Cas9 | A nuclease enzyme (protein) | Cuts both strands of the target DNA |
| Guide RNA | About 20 nucleotides matching the target | Leads Cas9 to the right place by base pairing |
| PAM | A short DNA sequence beside the target | Must be present for Cas9 to cut |
| End-joining repair | The cell rejoins the cut ends, often imperfectly | Small insertions or deletions knock the gene out |
| Template repair | The cell copies a DNA template supplied with Cas9 | A precise change or an inserted sequence |
Watch out for (5)
- Transgenic DNA can cross any kingdom→ Genetically modified organisms and their products
- The added allele is dominant, and it fades with the cells→ Gene therapy: somatic and germline
- Pluripotent is one step below totipotent→ Stem cells and potency
- A clone is not a perfect copy of the nucleus donor→ Cloning: natural, plant and animal
- The guide RNA aims; Cas9 cuts→ CRISPR-Cas9 gene editing in outline
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