Genetic engineering and CRISPR

Recombinant DNA

Genetic engineering means changing an organism's DNA directly in the lab. One way is to take a gene from one organism and put it into another. DNA that combines pieces from different sources is called recombinant DNA, and an organism carrying a gene from another species is transgenic. It works because the genetic code is almost universal (1D).

The classic example is making human insulin in bacteria:

1. The human insulin gene is cut out using a restriction enzyme, leaving sticky ends. (In practice, a copy of the gene is usually made from the mRNA, because human genes contain stretches that bacteria can't process.)
2. A plasmid, a small loop of DNA from a bacterium, is cut open with the same restriction enzyme, so it has matching sticky ends. The plasmid is the vector: the vehicle that carries the gene into the cell.
3. The gene and plasmid are mixed. Their sticky ends pair up, and the enzyme DNA ligase seals the backbones, making a recombinant plasmid.
4. Bacteria such as E. coli take up the plasmid. Those that have it are selected, often using an antibiotic-resistance gene also on the plasmid.
5. The transgenic bacteria are grown in huge steel fermenters. Every time they divide they copy the plasmid, and each cell makes human insulin, which is extracted and purified.

Human insulin from bacteria went on sale in 1982, the first medicine made by genetic engineering. Since then, recombinant DNA has been used to make growth hormone, clotting factors for haemophilia, the hepatitis B vaccine, and the enzyme chymosin used to make much of the world's cheese.

Genetically modified crops

To get a gene into a plant, scientists often use a soil bacterium, Agrobacterium, which naturally inserts some of its own DNA into plant cells. Whole plants are then grown from the transformed cells. Examples include:

• Bt cotton. Genes from the soil bacterium Bacillus thuringiensis make proteins that are toxic to caterpillars such as cotton bollworms, but harmless to people, mammals and most other insects. GM cotton was first grown in Australia in 1996 and now makes up over 99% of the Australian crop. The CSIRO estimates it has cut insecticide spraying on cotton by about 85%. Farmers must also plant "refuge" crops without the Bt gene, to slow the evolution of resistant pests.
• Herbicide-tolerant canola, grown in several Australian states, lets farmers control weeds with fewer, more targeted sprays.
• Golden Rice makes beta-carotene, which the body turns into vitamin A. Vitamin A deficiency causes blindness in hundreds of thousands of children a year in poor countries. Golden Rice was approved for growing in the Philippines in 2021, though legal challenges have since held it up.
• Drought-tolerant wheat. Food Standards Australia New Zealand has approved food from HB4 wheat, which carries a sunflower gene that helps it cope with drought.

In Australia, the Office of the Gene Technology Regulator decides whether a GMO can be released, and Food Standards Australia New Zealand assesses GM foods for safety and requires most to be labelled.

Reading DNA: sequencing and bioinformatics

To engineer genes you need to know their sequence. In 1977 Fred Sanger developed a method of reading DNA one base at a time by copying it with nucleotides that stop the copy at each A, T, G or C. It earned him his second Nobel Prize. Sanger sequencing was used for the Human Genome Project and is still used to check short stretches. Since about 2007, next-generation sequencing machines read millions of short fragments at the same time, and newer machines can read single DNA molecules tens of thousands of bases long, some by threading DNA through a tiny pore in a membrane. The cost of a human genome has fallen from about US$100 million in 2001 to a few hundred dollars (1B).

Such machines produce far more data than a person could read. Bioinformatics uses computers to assemble sequences, find genes, compare genomes and predict protein shapes. In 2020 the artificial intelligence program AlphaFold, from Google DeepMind, predicted the 3D shapes of proteins from their amino acid sequence with remarkable accuracy, and its creators shared the 2024 Nobel Prize in Chemistry with American biochemist David Baker, who designs new proteins by computer.

CRISPR: editing genes

Earlier methods added genes at random places. Gene editing changes a chosen base sequence at a precise location. The most important tool, CRISPR-Cas9, was borrowed from bacteria. Bacteria store short pieces of DNA from viruses that have attacked them in a region of their DNA called CRISPR. They make RNA copies of these pieces and load them into an enzyme, Cas9, which patrols the cell and cuts any DNA that matches, destroying the virus. In 2012 French microbiologist Emmanuelle Charpentier and American biochemist Jennifer Doudna showed this system could be reprogrammed to cut any DNA sequence chosen. They won the 2020 Nobel Prize in Chemistry.

• Scientists make a guide RNA with about 20 bases that match the target gene.
• The guide RNA leads Cas9 to the matching DNA. Cas9 only cuts if the target sits next to a short sequence called the PAM (for the usual Cas9, NGG: any base then two Gs).
• Cas9 cuts both strands of the DNA.
• The cell repairs the cut. Usually it joins the ends roughly, adding or losing a few bases, which knocks out (switches off) the gene. If scientists add a piece of DNA with the desired sequence, the cell can use it as a template to write in a precise change.

CRISPR is cheap, fast and works in almost any organism, so it is now used in labs around the world: to find out what genes do by switching them off, to develop disease-resistant crops, and to treat disease. Newer versions called base editors and prime editors can change single bases without cutting both strands.

Gene therapy

Gene therapy treats a genetic disease by adding a working gene to a patient's cells, or editing their own. The first trial was in 1990, on a four-year-old American girl with an immune disease, ADA deficiency. For the next 25 years progress was slow: getting genes into enough of the right cells proved hard, the patient's immune system often attacked the viral vectors used to carry the genes, and in 1999 a trial participant died. Cystic fibrosis gene therapy, widely expected to succeed in the 1990s, has still not worked well enough to be used (1E).

Since 2017, gene therapies have become real treatments:

• Luxturna (approved in the USA in 2017) delivers a working copy of a gene into the retina, restoring some sight in a rare inherited blindness.
• Zolgensma (2019) treats spinal muscular atrophy, which used to kill most affected babies before age two, with a single infusion.
• Casgevy (approved in the UK and USA in late 2023) was the first CRISPR treatment. Blood stem cells are taken from a patient with sickle cell disease or β-thalassaemia, edited to switch off a gene (BCL11A) that normally turns off foetal haemoglobin after birth, and returned to the body. The cells then make foetal haemoglobin, which doesn't sickle. Most patients in trials stopped having pain crises.

These treatments edit somatic cells, so the changes are not inherited. They also cost from hundreds of thousands to several million dollars per patient, raising questions about who can access them.