CRISPR-Cas9 Gene Editing (2012): Doudna and Charpentier

CRISPR-Cas9 gene editing is a method for cutting DNA at precise, chosen locations, first demonstrated in a paper by Jennifer Doudna, Emmanuelle Charpentier and colleagues published on 28 June 2012. Adapted from a natural immune system in bacteria, it made editing genes far cheaper, faster and easier than earlier techniques. Within a decade it had transformed biology and medicine and won its discoverers the 2020 Nobel Prize in Chemistry.

Background: a bacterial immune system

The story began with odd patterns in microbial DNA. In 1987 Japanese researchers led by Yoshizumi Ishino noticed unusual repeated sequences in the bacterium E. coli. In the 1990s the Spanish microbiologist Francisco Mojica studied similar repeats in salt-loving archaea, and in 2002 the name CRISPR was proposed by Ruud Jansen and Mojica.

In 2005 Mojica and other groups noticed that the “spacer” sequences between the repeats matched viral DNA, and suggested that CRISPR was a form of acquired immunity. In 2007 researchers at the Danish food company Danisco confirmed this in bacteria used to make yogurt: the microbes stored snippets of viruses that had attacked them and used them to resist later infection.

These discoveries built on half a century of molecular biology since the discovery of the DNA double helix. Scientists already had tools for editing genes, such as zinc-finger nucleases and TALENs, but each new target required a newly engineered protein, a slow and costly process.

The 2012 breakthrough

Emmanuelle Charpentier, a French microbiologist then working at Umeå University in Sweden, studied the CRISPR system of Streptococcus pyogenes. In 2011 her group reported a small RNA molecule, tracrRNA, that was essential for the system to work. She met Jennifer Doudna, a structural biologist at the University of California, Berkeley, at a conference in Puerto Rico that year, and they agreed to collaborate.

Working with postdoctoral researcher Martin Jinek and Charpentier’s student Krzysztof Chylinski, the teams showed that the Cas9 enzyme, guided by two RNA molecules, cut double-stranded DNA at a site matching the guide. They then fused the two RNAs into a single “guide RNA” and showed that by changing about 20 letters of its sequence, Cas9 could be directed to cut almost any DNA target.

Their paper appeared online in the journal Science on 28 June 2012. It proposed that the system could be used for genome editing. At about the same time, the Lithuanian biochemist Virginijus Šikšnys reached similar conclusions, published in September 2012.

How the tool works

In bacteria, Cas9 is an enzyme that cuts the DNA of invading viruses. It is steered by RNA copied from the spacer sequences, so it attacks only DNA matching a past infection. A short additional sequence next to the target, called a PAM, tells Cas9 that the DNA is foreign and protects the bacterium’s own CRISPR store from being cut.

The 2012 paper showed that scientists could replace the natural guide with a synthetic one of their choice. Once Cas9 cuts both strands of DNA, the cell’s own repair machinery takes over. Often the repair is sloppy, adding or deleting a few letters and disabling the gene. If a DNA template is supplied, the cell can instead copy it into the break, allowing a precise change. The same Cas9 protein can be pointed at any gene simply by making a new guide RNA, which can be ordered cheaply from laboratory suppliers.

Rapid adoption

In January 2013 several groups, including those of Feng Zhang at the Broad Institute of MIT and Harvard and George Church at Harvard, showed that CRISPR-Cas9 could edit genes in human and mouse cells. Within months, laboratories around the world were using it in plants, animals and cell cultures.

The technique spread because it was cheap, simple and adaptable. Researchers developed variants that switch genes on or off without cutting, “base editors” that change a single DNA letter, and “prime editors” that rewrite short stretches. It accelerated work that had begun with the Human Genome Project, allowing scientists to test what individual genes do.

Doudna and Charpentier received many awards, including the Breakthrough Prize in Life Sciences in 2015. The Nobel Prize in Chemistry, announced on 7 October 2020, was given “for the development of a method for genome editing”; by then Charpentier was leading a research unit of the Max Planck Society in Berlin.

Ethics and controversy

The power of the technique raised serious ethical questions. In 2015 Doudna and others called for a pause on editing human embryos for reproduction, and an international summit discussed the issue in Washington that December.

In November 2018 the Chinese researcher He Jiankui announced that twin girls had been born from embryos he had edited to resist HIV. The work was widely condemned as unsafe and unethical, and a Chinese court sentenced him to three years in prison in December 2019. The case strengthened calls for strict rules on heritable human genome editing.

A long-running patent dispute also arose between the University of California, representing Doudna and Charpentier, and the Broad Institute. US patent rulings in 2017 and 2022 largely favoured the Broad Institute for applications in plant and animal cells.

By the numbers

Medical and agricultural uses

Gene editing has moved into medicine. In November 2023 the United Kingdom’s medicines regulator approved Casgevy, a treatment in which a patient’s blood stem cells are edited to restore production of fetal haemoglobin, for sickle cell disease and beta thalassaemia; the US Food and Drug Administration approved it for sickle cell disease in December 2023.

Other approaches edit genes directly inside the body. In 2021 researchers reported the first results of a trial in which CRISPR components were delivered to the liver by infusion to treat transthyretin amyloidosis, a rare inherited disease. Trials have also tested treatments for inherited blindness, high cholesterol and certain cancers, although many questions remain about long-term safety and cost.

Edited crops and livestock, such as disease-resistant plants, have been developed, though rules on their sale differ between countries. CRISPR-based tests have also been developed for detecting viruses. Debates over gene drives, which could spread edits through wild populations, continue.

Timeline

• 1987: Japanese scientists report unusual repeated sequences in E. coli DNA.
• 2002: The name CRISPR is proposed.
• 2007: Danisco researchers show that CRISPR protects bacteria against viruses.
• 2011: Charpentier’s group describes tracrRNA; she and Doudna begin collaborating.
• 2012, 28 June: The Jinek, Doudna and Charpentier paper is published online in Science.
• 2013, January: CRISPR is used to edit human and mouse cells.
• 2018, November: He Jiankui announces the birth of gene-edited twins.
• 2020, 7 October: Doudna and Charpentier are awarded the Nobel Prize in Chemistry.
• 2023, November: Casgevy becomes the first approved CRISPR therapy.

Why it matters

CRISPR-Cas9 turned gene editing from a specialist craft into a routine laboratory tool. It has opened new treatments for inherited diseases and new possibilities in agriculture, while forcing societies to decide how far humans should go in altering the genetic code. Like Dolly the sheep before it, it brought biotechnology to the centre of public debate.