Hello CRISPR
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To understand the origins of CRISPR, we have to go back to 1987, when a Japanese student discovered repeating sections of DNA in bacteria.
The student in question, Yoshizumi Ishino, was sequencing a gene called iap in E.Coli bugs. This is a straightforward task nowadays, but in the late 80s it was a complicated and time consuming process just to read the thousand or so letters of DNA that made up the gene in question. But eventually, he did it, publishing the sequence in the Journal of Bacteriology, when this kind of thing was still a big deal.
In the paper, Ishino noted that the gene seemed to contain repeating sequences of 29 base pairs, separated by unique sections of DNA, but he couldn’t figure out what they were doing and he didn’t pursue it further. His paper ends with the immortal words “So far, no sequence homologous to these has been found elsewhere in procaryotes, and the biological significance of these sequences is not known.”
The mystery of the repeating sections of DNA lay dormant for a few more years until they were spotted again in the early 1990s. This time it was Francisco Mojica, a student at the University of Alicante in Spain, who was studying archaea - single celled organisms that are very similar to bacteria, and also noticed the repeated sections of DNA. The fact that these repeating sequences appeared in two very different organisms - E.coli and archaea, convinced Mojica that they must have some kind of biological relevance.
By 2003, DNA sequencing had sped up considerably, and there were now databases containing the genetic sequences of an array of species. Curiously, these repeated sequences were turning up so often in microbes that they had even picked up their own name. In 2002, researchers in the Netherlands published a paper describing these distinctive repeats in 40 microbial species and proposing the term SPacers Interspersed Direct Repeats (SPIDR). But it was Mojica who came up with the name that stuck - Clustered regularly interspaced short palindromic repeats, or CRISPR for short. But it wasn’t enough to know that these repeats existed. Mojica wanted to know why they were there and what they did.
For years, Mojica tried to solve the mystery of the repeating sections of DNA, theorising that they may have something to do with cell division. Or maybe they helped to shape the structure of DNA? Or maybe they formed loops for proteins to attach to? Or maybe, I dunno, something else?? Much of the rest of the scientific community considered the repeats to be irrelevant ‘junk DNA’, but Mojica knew that bacteria and archaea didn’t have space to waste on useless genetic material within their sparse, compact genomes.
Mojica decided to delve deeper into the sections of DNA that lay between the CRISPR sequences in E.Coli. And when he did, he found that all these stretches of intervening DNA matched sequences from viruses that attacked bacteria. It turned out that bacteria that had incorporated sections of DNA from a virus in between some of their CRISPR repeats were immune to that particular virus, while those without became infected. Not only that, but the CRISPR system was adaptive: when a new virus came along, the bacteria incorporated some of the virus's DNA in between its CRISPR repeats. That’s when it hit him: maybe the sections of DNA and the CRISPR system formed some kind of bacterial immune system.
At first, the scientific community was sceptical about Mojica’s discovery. He sent his paper to the journal Nature, which said it wasn’t that interesting, and it took many frustrating months for him to get it published anywhere.
Still, soon a flurry of other publications confirmed his suspicions, and scientists worldwide became interested in the CRISPR system and how it worked.
When a virus infects a bacteria, CRISPR associated enzymes cut out a section of the viral DNA. The section, called a protospacer, is stored in the CRISPR section of the bacterial genome, between the repeated sections of DNA that Ishino and Mojica originally identified. If the virus infects the cell again, the bacteria use these sequences to defend themselves.
The stored sections of viral DNA are ‘read’, or transcribed, to generate short sections of RNA. These RNAs then team up with CRISPR associated DNA-cutting enzymes, known as CAS, to search the cell for viral DNA or RNA that matches that stored sequence. And if this CRISPR RNA-CAS complex finds a matching sequence from an invading virus, it makes a neat snip straight through the viral RNA or DNA, stopping the invader in its tracks.
The implications were huge: if the CAS ‘scissors’ could be directed to cut up any piece of DNA by altering that RNA CRISPR guide, then this was potentially the perfect programmable gene editing system that scientists had been searching for for so long. The only problem was actually getting their hands on it.
CRISPR comes to life
Although by 2010 scientists were beginning to understand how the CRISPR system worked, no one had isolated the molecular components of CRISPR, and we still didn’t understand how many of them functioned on a molecular level.
In 2011, Professor Jennifer Doudna from the University of California, Berkeley and Professor Emmanuelle Charpentier from the University of Vienna decided to collaborate, investigating one particular CRISPR associated enzyme called Cas 9.
They found that Cas 9 is what’s known as a dual RNA guided protein. It uses the stored sections of viral DNA transcribed into RNA molecules called crRNA to direct the Cas 9 enzyme to its target viral RNA, but it also needs an additional piece of RNA called tracrRNA, which helps bind the crRNA to the Cas 9 protein and activates the complex.
Soon what began as a curiosity-driven investigation became a project with broader implications. Once they understood how the dual RNA guides worked, they soon figured out that Cas 9 could be programmed with a single piece of RNA combining the roles of the crRNA and tracrRNA, directing the Cas9 complex to cut double-stranded DNA at a specific sequence.
After completing experiments proving they could successfully program Cas 9 to cut DNA at specific sites, Doudna and Charpentier filed a patent on the use of the CRISPR-Cas9 technology in genome editing. They also sent a paper covering their results to the journal Science, which fast-tracked its publication in June 2012. A second publication in January 2013 confirmed that the system also worked in human cells.
The beauty of CRISPR gene editing lies in its simplicity. RNA plus Cas9 is a simple system of precision guided genetic scissors that can be programmed to cut any sequence of DNA. And once that cut has been made, nature takes its course, patching up the break with any DNA nearby that happens to match the broken ends through homologous recombination. And if you also provide a matching DNA repair template, complete with any variations or changes you want to incorporate, then those changes will be pasted back into the gene. And there you have it: genome editing.
You’d think that would be the end of our dramatic story about the discovery of CRISPR, but in truth, the drama was only just beginning.
Who owns CRISPR?
Doudna and Charpentier, although the first to publish their results and widely credited as the discoverers of the CRISPR Cas-9 system, were not the only team working on it.
A Lithuanian molecular biologist called Virginijus Siksnys also submitted a paper with similar results to Doudna and Charpentier to Cell in April 2012, but his manuscript was rejected without review, then rejected again by Cell Reports before it was eventually published in the Proceedings of the National Academy of Sciences (PNAS), in September 2012, months after Doudna’s paper.
The delay in publishing meant that Siksnys’s work was largely overlooked, and his name is rarely mentioned in the CRISPR conversation. However, he did take a share of the Kavli Prize in nanoscience in 2018 together with Doudna and Charpentier.
Meanwhile, in 2012, bioengineer Professor Feng Zhang from the Broad Institute at MIT and geneticist Professor George Church from Harvard independently showed that the CRISPR Cas 9 system could be used to edit DNA in human cells. They also filed several patents on their discoveries, explicitly covering the ability of CRISPR-Cas9 to edit eukaryotic cells in December 2012. Their institutions paid to expedite the review of their patent, so they were approved first in April 2014, even though their applications were filed after Doudna’s.
The University of California and University of Vienna team representing Doudna and Charpentier petitioned for patent interference. Unfortunately, Doudna’s patent only presented evidence for the use of CRISPR in prokaryotic bacterial cells, while Zhang’s presented specific evidence for its use in eukaryotes, including humans.
Doudna’s attorneys argued that CRISPR could obviously be used in eukaryotes too, with no ‘special sauce’ required for the move, and the team from the Broad institute simply followed the instructions published in their paper. But Zhang’s team argued that the work published by Doudna and her team was not enough to enable CRISPR to be used in eukaryotes, and additional ‘inventiveness’ was required.
Of course, this urgent need for each team to put their stamp on CRISPR was a reflection of the potentially vast sums of money at stake for whoever got to claim ownership of the technology, and therefore license it out to anyone who wants to use it. Then things took an even more unpleasant turn.
In 2016, the president and founding director of the Broad Institute, Eric Lander, published an article in the journal Cell called “The Heroes of CRISPR”. It focused almost entirely on the men in the story, particularly Zhang, the ‘hero’ responsible for exploring the use of CRISPR in human cells, while downplaying the roles of Doudna and Charpentier.
After its publication, many accused Lander of trying to weave a new historical narrative and control the optics around the patent dispute. Men trying to override the scientific contributions of women? Surely not...
In February 2017, the patent office ruled against Doudna and Charpentier, saying that eukaryotic CRISPR was a separate technology from other applications. Doudna’s team took the issue to the federal court, but their appeal was denied in 2018. And in 2019, they filed another patent interference claim, which is still ongoing.
Beyond the patent war, Doudna and Charpentier did get recognition for their work in the form of the 2020 Nobel Prize in Chemistry, becoming only the sixth and seventh women ever to win the prize, and the first women to win without male co-winners.
However, as always, some are unhappy, especially as a Nobel can be awarded to up to three people. Many scientists contributed to CRISPR, with obvious third candidates including Moijca, Zhang, or even Siksnys. But perhaps after all these years of women being passed over for the prize, the committee wanted to show what it felt like to be the other way round for once?
References:
The Code Breaker - Jennifer Doudna, Gene Editing, and the Future of the Human Race. Walter Isaacson
CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants
Francis Mojica - the modest microbiologist - Technology Networks
These Scientists Deserved A Nobel Prize, But Didn’t Discover Crispr - Forbes
Pioneers of revolutionary CRISPR gene editing win chemistry Nobel - Nature
Nobel Prize for CRISPR honors two great scientists – and leaves out many others - The Conversation
Image Credit:
Jennifer A. Doudna. © Nobel Prize Outreach. Photo: Brittany Hosea-Small.
Emmanuelle Charpentier. © Nobel Prize Outreach. Photo: Bernhard Ludewig.
