THE citation for the year’s Nobel Prize in Chemistry sounds rather trite, merely saying that the prize was awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing”. It hardly conveyed the enormous power of the genetic technique the laureates had discovered nor its wide-ranging implications. The press release and the background document the Royal Swedish Academy of Sciences put out did somewhat better. Emmanuelle Charpentier and Jennifer Doudna, they said, had discovered one of gene technology’s sharpest tools—the CRISPR/Cas9 “genetic scissors”—which researchers can use to change the DNA of animals (including humans), plants and microorganisms with extremely high precision. “Using the CRISPR/Cas9 genetic scissors,” the press release added, “it is now possible to change the code of life over the course of a few weeks [emphasis added].”
Fifty-two-year-old Emmanuelle Charpentier is a French researcher in microbiology, genetics and biochemistry and is currently at the newly established Max Planck Unit for the Science of Pathogens in Berlin, Germany. Fifty-six-year-old Jennifer Doudna is an American biochemist currently at the University of California in Berkeley (UCB), United States. They are the sixth and seventh female laureates in chemistry, which takes the total number of female laureates in chemistry to about 4 per cent. This is the first time that two women have been jointly awarded the Nobel Prize. The two were otherwise basically engaged in different areas of biology: Emmanuelle Charpentier studying bacterial systems and Jennifer Doudna RNA biology. They came together in, according to Emmanuelle Charpentier, a “brief but intense” collaboration after a meeting in a café in Puerto Rico during a conference. Their joint objective was to specifically study the CRISPR machinery in a particular Streptococcus bacterium known to cause the greatest harm to humanity, which Emmanuelle Charpentier had been researching into for some years.
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The power of the technique and the ease with which a reasonably well-skilled bio-researcher can deploy it to change the genome of any organism make the italicised part above literally true, which naturally gives rise to a whole lot of ethical questions and issues about how use of this technology should be regulated while not denying humanity the potential tremendous benefit it promises. The technology has had a revolutionary impact on molecular life sciences and is already contributing to potential cancer therapies and, as the press note observed, “may make the dream of curing inherited diseases come true”.
For instance, it has already undergone clinical trials to test whether it can cure blindness and sickle-cell anaemia. It has been used to create gene-altered plant and crop varieties with additional beneficial attributes such as drought resistance. Within six years of the laureates’ discovery of the technique, there have been, on the one hand, patent disputes and law suits with regard to the technique’s applicability to human cells and, on the other, a controversial application in in vitro fertilisation (IVF) to create designer babies. Sure enough, following the concern and uproar in the scientific community, the law has caught up with the latter case. A little more on this later, but first the basic science for which the laureates have been awarded.
Simply stated, the new genome-editing CRISPR/Cas9 system is a molecular complex that was discovered in prokaryotes—lower order organisms such as bacteria and archaea whose cells do not have an enveloped nucleus—as part of their evolutionarily built-in and well-preserved adaptive immunity system to protect them from invading bacteriophages (viruses that infect bacteria) and plasmids. Bacteria use the system to recognise invading pathogens and carve their DNA up to make them non-functional and non-infective. From the 1990s, when the Spanish molecular biologist Francisco Mojica came out with a full characterisation of what is now called CRISPR, to about 2011, research on the CRISPR system was essentially around its workings in various prokaryotic biological systems.
In June 2012, Emmanuelle Charpentier and Jennifer Doudna published the ground-breaking paper that showed how this natural immunity machinery of lesser species could be converted into a “programmable” and easy-to-use editing tool to cleave the DNA of any organism (most importantly of eukaryotes, such as plants and animals, whose cells have enveloped nuclei) at any predetermined site. So these “search-and-replace”, or “cut-and-paste” if you like, genetic scissors enable a biotechnologist to cut up any genome at points of interest, throw away undesirable (say, disease-causing) mutations in it and replace them with desirable ones or just insert any genes carrying desirable additional trait(s). That is, where the DNA is cut, the “code of life” can be rewritten. The possibilities thus become mind-bogglingly endless and, if unregulated, even scary.
The laureates’ discovery was entirely unexpected. The two did not set out to actually devise a pair of genetic scissors. They had, in fact, started investigating the immune system of the bacterium Streptococcus pyogenes, and one of their ideas was to see whether they could develop a new kind of antibiotic. The possibility of a gene editor based on the CRISPR/Cas9 system just presented itself to them during their collaborative work. It has been said about Emmanuelle Charpentier that she always looks for the unexpected; quoting Louis Pasteur, she herself has said: “Chance favours the prepared mind.”
The story of the work that led to the discovery of CRISPR/Cas9 goes back to 1987 when the Japanese researcher Yoshizumi Ishino noted the presence of unusual sequences repeated in the genome of the organism Escherichia coli that were interspersed by different spacer sequences. That is, distinct, identical and perfectly conserved sequences were separated at regular intervals by unique sequences that varied. It is like the same word being repeated between each unique sentence in a book. Later, Mojica also showed that similar structures are to be found in the genome of the (halophilic) Archaea Haloferax mediterranei, which is found in extreme salty environments.
The acronym CRISPR
Subsequent research using bioinformatics analysis revealed that these types of repeats are common in prokaryotes with very similar features: a short, palindromic element occurring in clusters and separated by unique intervening sequences of constant length. The highly conserved and preserved nature of these structures suggested that they were of ancient evolutionary origin and had high biological relevance. In 2002, Ruud Jansen of Utrecht University in the Netherlands coined the acronym CRISPR for clustered regularly interspaced short palindromic repeats. An important element in the understanding of the function of CRISPR was the finding around 2002 by Jansen and associates of CRISPR-associated (Cas) genes. This group of genes was found only in prokaryotes that contained the CRISPR structures and were always located near CRISPRs. The proteins encoded by these genes suggested that Cas genes had a role in DNA metabolism and gene expression. Subsequently, many Cas proteins were identified, but the functional significance of CRISPR and the associated Cas genes remained elusive.
Around 2005, researchers found that the unique, non-repetitive sequences of CRISPR seemed to match the transmissible genetic code contained in bacteria-infecting pathogens such as bacteriophages and plasmids. This correlation seemed to suggest—and indeed it was found to be so—that these sequences were evolutionarily derived from these pathogens, and prokaryotes carrying these sequences seemed to be protected from being infected by the pathogens.
The current hypothesis posits that if a bacterium has successfully survived any invasion from these pathogens, it integrates the relevant genetic code into its genome as a memory of past infection. The hypothesis was experimentally verified in 2007 by infecting a strain of S. thermophilus with virulent bacteriophages. The experiment revealed that the resistant bacterium had acquired new spacer sequences in its genome. The exact mechanism of how all this works was yet to be fully understood, but the knowledge already gathered was sufficient to make further progress in the direction that ultimately led to the Nobel Award–winning discovery.
What research had already shown is that CRISPR sequences in the bacterial genome were transcribed into long RNAs, which were then cleaved from within (by the enzyme RNase III) to yield small RNA elements called CRISPR-RNAs (crRNAs). This led to the conjecture that crRNAs in bacteria played a role in targeting viral proteins perhaps in a manner similar to the mechanism known as “RNA interference”. This is a naturally occurring mechanism by which an organism uses endogenously generated short strands of RNA to interfere with the expression of certain targeted genes and thereby suppress or silence them. It was also later demonstrated that crRNA formed molecular complexes by binding with one or more Cas proteins that targeted invading pathogenic proteins (Figure 1).
In 2006, when the discoverers of RNA interference were awarded the Nobel Prize in Physiology or Medicine and the field itself was in an exciting phase of development, Jennifer Doudna, with over two decades of experience in working with RNA and now leading a research group at the UCB, had just begun to do research in the field of RNA interference. It is then that she learnt from a colleague in another department about the discovery of the CRISPR-Cas system and the machinery’s similarity to RNA interference. Jennifer Doudna was excited by this and the fact that Cas genes are very similar to genes that code for already known proteins that are involved in unwinding and cutting DNA. She set down to find answers to whether Cas proteins had the same function and could also cleave the viral DNA.
In a few years’ time, her research group succeeded in revealing the functions of several different Cas proteins. Studies with the CRISPR/Cas systems in other research laboratories and universities revealed that there were many CRISPR/Cas systems depending on the Cas protein(s) that they coded for and that these could be divided into two broad classes: Class 1 systems in which many Cas proteins combined to form large CRISPR-associated molecular complexes to mount a bacterial defence against invading viruses and the much simpler Class 2 systems in which molecular complexes were formed with much fewer proteins, or even just a single multifunctional Cas protein such as Cas9, to effectively prevent viral proteins from being expressed. The CRISPR/Cas system that Jennifer Doudna’s group had been working with belonged to Class 1, whereas the system that Emmanuelle Charpentier was working with in S. pyogenes belonged to the simpler Class 2.
Emmanuelle Charpentier began her work with S. pyogenes in 2002 when she started her own research group at the University of Vienna. In trying to understand the extreme infectivity of the bacterium, which affected millions of people every year and in some cases could turn life-threatening, she began with a thorough study of how its genes are regulated. By the time she moved to Urneå University in north Sweden in 2009, she, in collaboration with researchers in Berlin, had already mapped all the small, gene-regulating RNA molecules in S. pyogenes. One such small RNA molecule, which was found in large amounts in the bacterium, had intrigued her. For one, this was an as yet unknown variant and, secondly, its genetic code was very similar to the repeating CRISPR sequence in the bacterium’s genome, leading her to suspect that the two must be linked. Indeed, her careful analysis confirmed that the genetic code of one part of the small unknown RNA molecule matched the part of the CRISPR that was repeated.
Although she had never worked with CRISPR before, she initiated a complete mapping of the CRISPR system in S. pyogenes, which was already known to belong to Class 2 as it required only Cas9 to cleave the viral DNA. Emmanuelle Charpentier demonstrated that the unknown RNA molecule was an integral part of the CRISPR-Cas system of S. pyogenes and that it had a decisive function. She showed that the RNA molecule, which she termed trans-activating crispr RNA (tracrRNA), was necessary for the long RNA molecule created from the CRISPR sequence to mature into its active form (Figure 1). After extensive experimentation, Emmanuelle Charpentier published her discovery of tracrRNA in March 2011. She had a hunch that she was on to something exciting. Unlike much of research, which is often not clearly black or white, here it was really white, she said in her telephonic interview with the Nobel Media soon after the announcement of the award. So, to continue her investigations on the CRISPR-Cas9 system in S. pyogenes, she felt the need to collaborate with a biochemist, and the natural choice was Jennifer Doudna.
On her choice of Jennifer Doudna as her collaborator, Emmanuelle Charpentier said: “[It was] a wish from both sides, and an understanding that we needed to go fast because… the story was a great one, so that’s why it was intense—it was a common understanding that it was important to join forces… and be fast…. this is also part of the reason why I approached Jennifer Doudna… we were very much in line in the way to do very precise research…. it was fast but precise, and deep. For this we recognised one another—we are the same type of scientist who… want to see the details of the data, so this was… important because… this is not about a paper published in… the high impact-factor journals. It’s really about… solid work.”
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By coincidence, Emmanuelle Charpentier got a chance to meet Jennifer Doudna at a conference in Puerto Rico where the former had been invited to speak about her work and discuss the possibility of collaboration, and the joint work took off from there. Their starting point was a suspicion that in the case of S. pyogenes the long RNA read-out from CRISPR was needed to identify a viral DNA and that Cas9 was the bacterial scissor that cut off the DNA molecule. But when they tested this hypothesis in vitro, nothing happened. The DNA remained intact. Following a great deal of discussion and many failed experiments, the duo decided to add the tracrRNA molecule that Emmanuelle Charpentier had discovered. Voila! The DNA molecule was cleaved into two parts. Emmanuelle Charpentier had thought that tracrRNA was only needed to activate the CRISPR-RNA once it was cleaved. It now became clear that tracrRNA had a crucial role to play in bacterial defence.
Leap of imagination
Having fully uncovered the fundamental mechanism in the bacterial defence against invading pathogens, the duo could have ended their joint work there. But then came the leap of imagination. They decided to create a new single molecular complex by combining crRNA and tracrRNA to form a kind of “guide RNA molecule” and see whether it could be used as a general purpose genetic tool that could cleave any genetic element of choice not just that of viral DNA.
Using a gene that was already there in a freezer in Jennifer Doudna’s laboratory, the duo selected five sites for cleaving. Accordingly, they altered the CRISPR spacer sequences to match the genetic codes of the sites where they desired the cuts to be made. And this worked like magic. The guide molecule carried Cas9 protein to the gene sites where incisions had to be made, and the gene was cleaved exactly at the five identified sites. The finding turned out to be the Nobel Prize–winning magical genetic tool (Figure 2).
“I think we had a sense in those very early days,” Jennifer Doudna said in her post-announcement telephonic interview, echoing Emmanuelle Charpentier’s words, “that… we were onto something big, but I think we had no idea how big. And it still amazes me every day to see the extraordinary work that’s going on now globally with this technology… and thinking back about how it really started with just a curiosity driven project… and how much more they [bacteria], I am sure, still have to teach us.”
The work of many researchers paved the way for this discovery, and the fact that only two winners were chosen when the rules allow a Nobel Committee to go up to three has generated some controversy. The most notable miss, according to experts, is Virginijus Šikšnys, a Lithuanian biochemist at the University of Vilnius who made similar discoveries. Indeed, Šikšnys is supposed to have sent his work for publication earlier to a well-known, highly respected journal only to have it rejected.
Because of the simplicity of the method, the field of CRISPR-based research, the technique’s applications and related technology have really exploded, and there is even a journal dedicated to the field. Because all that is involved in the technique is one specialised DNA-cutting (Cas) protein and a guiding molecule, the next step was obvious: to see whether the magical scissors could also work on the genes of human cells. In January 2013, the work of George Church and Feng Zhang of Harvard answered the question in the affirmative. A few weeks later, Jennifer Doudna, too, published similar results from her laboratory. Zhang, who has already got his finding patented, is now involved in a costly dispute over CRISPR patent rights with Emmanuelle Charpentier and Jennifer Doudna.
While the technology of CRISPR/Cas9 genetic scissors has been hailed, the biggest biotechnology discovery of the century, as mentioned earlier, is the technology’s potential for controversial uses both in research and in applications in medical technology, particularly when it comes to editing human genomes. In 2018, a Chinese scientist, Jian-kui He, used it on IVF embryos to create the first babies with genome editing. His work attracted widespread condemnation, and he is now serving a three-year prison sentence followed perhaps with a lifetime ban on his pursuing biology research.
Emmanuelle Charpentier stated in her interview: “CRISPR-Cas has facilitated a lot, and genetics in research and development. But as to have it as a technology that can be used safely for the editing of the human germline it’s something else, first of all. And second of all one should not underestimate… that CRISPR/Cas9, even though it is a wonderful tool, it would be extremely difficult to get the technology to modify more than one gene at a time. So, I think… we may see unfortunate and really unwanted experiments.” In this regard, Jennifer Doudna has taken on a public role with initiatives to warn society about the potential misuse that would seem imminent unless tight regulations are put in place at the global and individual country level. The WHO has already come out with guidelines (as determined by a special panel) on regulating the technology.
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