It all started in the freezer of an anonymous laboratory belonging to the University of Alicante. It was the hot summer of 1993 when the microbiologist Francisco Juan Martínez Mojica patiently analyzed the genetic material of the archaea Haloferax mediterranei , a unicellular microorganism collected “God knows when” – according to his words – in the Alicante salt flats of Santa Pola. He quickly realized that those frozen samples had very peculiar characteristics. Repeated sequences of nucleotides, the molecular building blocks that make up DNA and RNA, appeared at regular intervals in the archaea genome .
Those were good times for those who were dedicated to studying the peculiarities of genomes. Just two years after Mojica discovered those inexplicable patterns, the first complete genetic instruction book for an organism was obtained. And, in subsequent years, sequences of both bacteria and archaea accumulated, both microbes without nuclei, but with different characteristics.
Thanks to the new flood of information, Mojica was able to investigate whether the repetitions that he had found in Haloferax mediterranei – a halophilic microbe, that is, that proliferates in very saline environments – were also presented by other species. And, to his great surprise, it did not take long to ratify it.
Those since then baptized as grouped and regularly interspaced short palindromic repeats – or CRISPR , for its acronym in English – and the spacer sequences – the genetic information found in the spaces that mediate between these repeats – began to serve as tools to identify strains of bacteria , a process known as spoligotyping. But for Mojica this was not enough. “I sensed that they had to fulfill an important function, because many cells died when we manipulated them,” he commented in an interview in El Confidencial .
In his search for answers, he turned to databases. It was a work of years that ended up paying off. “We finally found a spacer sequence in a strain of Escherichia coli bacteria identical to that of a sequence of a bacteriophage virus that infects different strains of said microbe. To verify that it was not something fortuitous, I located and made comparisons of the spacers present in the genomes of all prokaryotes –microorganisms without nuclei– where we were able to detect CRISPR regions … Et voilà, we found other coincidences ”.
It happened in 2003, a decade after the CRISPR sequences were discovered. Finally, Mojica realized what he was seeing: when bacteria had spacer sequences identical to fragments of the genome of certain viruses, these pathogens did not appear in microbes. I mean, they had become immune to his infection. Mojica had just discovered a primitive, adaptive, and also genetically based defensive system ; a kind of autovaccine that protects bacteria against viral attacks. And unlike our immune system, which must be educated to provide effective protection, it was heritable.
However, as often happens when we talk about drastic paradigm changes, the Alicante microbiologist suffered to see his findings published. The journals with the greatest impact rejected the manuscript until, in 2005, the lesser-known Journal of Molecular Evolution agreed to publish the article, after a long review process. Two years later, scientists at a Danish yogurt company called Danisco provided empirical confirmation that their conclusions were correct.
As Lluís Montoliu, a CSIC researcher, masterfully summarizes in his book Editing genes: cut, paste and color , “CRISPR systems are made up of long rows of repetitive sequences, interspersed by spacers that represent fragments of virus genomes or plasmids –small circular DNA molecule – that have previously attacked the microorganism. These sequences and spacers are transcribed, that is, they are copied and converted into RNA molecules, which remain inside the bacteria waiting to find the homologous DNA sequence with which to pair.
When the same virus infects the bacterium again, it recognizes the new infection, by pairing the small RNA molecule with its corresponding complementary sequence of the invading virus. The union takes place thanks to a protein, Cas – an acronym for English CRISPR associated protein -, which is capable of presenting RNA to its complementary DNA. Upon completion of mating, the microbe interprets that the same virus wants to re-enter. The Cas protein then activates its role as an endonuclease, an enzyme that cuts DNA internally at very precise positions. These sections in the double strand of viral DNA cause its degradation and decomposition ”.
You may be wondering: but how does it affect me? It was not until the summer of 2012 that this information traveled the tortuous path that leads from basic to applied research. From the pages of the journal Science , an article that will remain forever in the annals of the history of science informed the scientific community of the incredible potential that this selective DNA cutting mechanism had .
Signed by researchers Jennifer Doudna, a molecular biologist at the University of California at Berkeley (USA), and Emmanuelle Charpentier, current director of the Max Planck Institute for Infectious Biology in Berlin, the article proposed for the first time the use of the components of the CRISPR system – in this case, from the bacterium Streptococcus pyogenes – as a tool for gene editing. It is what later came to be called genetic glue cutters or molecular scissors .
The desire to manipulate and modify the genome has been with us since genetic studies have existed. Since the 1970s, scientists have experimentally turned genes on and off , but despite the therapeutic promises that have always accompanied discoveries in this field, the track record of success was disappointing. Until CRISPR / Cas9 arrived, as the system presented by Doudna and Charpentier is known.
This technique, the fourth in a series of methods based on the action of nuclease enzymes, constituted a qualitative leap so relevant that it earned its creators the Nobel Prize in Chemistry in 2020 . While to work with the TALEN, the predecessor tools, almost a hundred reagents were needed – chemical compounds that produce reactions – for the CRISPR, two are enough. Experiments that used to cost 5,000 euros – with poor results – can now be carried out for less than sixty. In addition, the paste-cut technique allows you to easily manipulate any region of the genome and even several areas at the same time.
Today, if we type ‘CRISPR’ in an Internet search engine, more than one hundred million results will appear. “He surpasses Leo Messi –65.3 million entries– and Cristiano Ronaldo –74 million–”, he acknowledges with amazement in his book Montoliu. According to the Global CRISPR / Cas9 Market Outlook 2022 report, it is estimated that the global market related to the new technology will exceed 1.5 billion dollars within three years . Thousands of scientific articles have been published in this regard and, as Montoliu defends in his conferences, “the limit of the applications is in the imagination of the researchers” .
With CRISPR / Cas9 it is possible to inactivate, modify or delete a gene or correct a mutation with a precision never seen before. But there’s a problem: For cells, breaks in DNA strands are traumatic events that can rarely be repaired without causing permanent damage.
Although all cells are equipped with systems to restore the integrity of the genetic material affected by an attack, their abilities are not as effective as we would like, and many times they give rise to mutations. This is very good if our objective is to inactivate a gene – or destroy an aggressor, as bacteria want – while if we want to play as we please with the genome, things get complicated. To overcome this limitation, the scientists add another sequence to the guide RNA, the tips of which correspond to those that would originate after cutting. Thus, the additive serves as a template for DNA double strand repair and there is a greater chance that scientists will integrate the new information into the cell.
The downside is that the alternative repair route is much more difficult to activate than the normal one, that is, the introduction and elimination of nucleotides like crazy until we find a combination capable of closing the fracture in the DNA molecule. This method, although effective, is anything but precise, and it also lacks memory: although there is a similar fracture in different cells, the information is not shared. As a consequence, the repair is done differently in each case. This fact gives rise, for example, to the so-called mosaic mice, with cells whose genomes show differences due to the various solutions carried out by the repair mechanisms.
The list of contraindications does not end there. CRISPR / Cas9 also has its own defects –not attributable to cellular healing mechanisms– that cause what are known as off-target mutations , translatable as “off-target”. Although the Cas9 endonuclease enzyme preferentially cuts at the sites where the RNA guide and the gene to be edited fit one hundred percent, there are other sequences in the genome whose differences in relation to said guide are minimal and that, many times, also they end up taking a bite. To overcome the problem, scientists play with the number of interventions and the time spent on them. By limiting both factors, you can fine-tune the precision and decrease the likelihood of off-target mutations.
All these obstacles do not prevent CRISPRs from being revolutionary and have allowed, for example, the rapid creation of gene-edited mice. If previously it took 12 to 18 months to develop a single mutant, Rudolf Jaenisch’s laboratory at the Whitehead Institute for Biomedical Research (USA) reduced that time to between four and six months thanks to CRISPR. And, in addition, they managed to introduce several mutations simultaneously.
As Montoliu recounts in his book, “nowadays, mutating a mouse gene has become a trivial, routine technique, within the reach of any laboratory minimally equipped with molecular biology and embryology techniques […]. Everything that has come after has been a torrent of information; thousands of publications that refer to the success of CRISPR tools to edit genomes of practically any imaginable organism ”.
And what about humans? Here we enter marshy terrain. Compared to previous tools, the CRISPR / Cas9 system has managed to increase the percentage of correctly repaired DNA molecules from 5% to 30%, but a risk of 70% is still unacceptable. It doesn’t seem like enough of an obstacle. According to Montoliu, “from the moment that CRISPR technologies became a reality, the possibility of editing the genome in human embryos and, with it, influencing their development and final characteristics, was too tempting.” What happened was a matter of time.
So it was. On April 18, 2015, Chinese scientists published the first study documenting this type of intervention in the journal Protein Cell. The biologists used leftovers from assisted reproduction protocols, which were also triplonuclear, that is, they resulted from the fertilization of an egg by two sperm. These were unviable embryos that could never give rise to a person.
After that, you just had to wait for the bomb to go off. At the end of 2018, He Jiankui, another Chinese scientist, revealed that he had used CRISPR to edit human embryos that he had subsequently implanted in a woman. Two twins had been born from that pregnancy; the scandal was huge.
Today much remains to be explained. Following the publication of a story about it in the MIT Technology Review , He had to reveal the details of his trials. Just before the presentation he made at the Second International Summit on Human Genetic Editing, where he had planned to announce his exploits, the biophysicist spoke of his experiments through videos on his own YouTube channel. His presentation, a tense 20 minutes to which he attended under escort, was confusing and left many unknowns. Hounded by journalists and fellow professionals alike, He ran right out of the box. Until 2019 no more was heard from him. On December 30 of that same year, the official Chinese news agency, Xinhua, announced that He Jiankui had been sentenced to 3 years in prison. Its results have not been published.
However, MIT Technology Review agreed to a manuscript that could have been submitted for publication to the journals Nature and JAMA (the Journal of the American Medical Association). According to the article, the objective of the experiment was to inactivate the CCR5 gene, which directs the synthesis of the protein that acts as a gateway for the AIDS virus into lymphocytes, to make the girls resistant to HIV . But, although the text affirms the “success” of this “novel therapy”, according to the experts consulted by MIT Technology Review , it hardly attempts to demonstrate this result, which, in addition, the data does not support.
We do not know if genetic surgery generated unwanted mutations in the babies, but specialists point out that the information released suggests that the editing was wrong. The scientific community has harshly criticized the experiment and some researchers comment that a Pandora’s box has been opened. Proof of this is that a Russian biologist, Denis Rebrikov, now intends to reproduce the Chinese scientist’s experiments and try to eliminate the GJB2 gene, related to deafness, in an embryo.
In Spain, a signatory to the Asturias Convention that prohibits including genetic modifications in the human genome that can be transferred to the offspring, carrying out a trial of this type would be illegal. Furthermore, considering the current development of the technique, it is not difficult to conclude that such interventions would be premature and entirely unsafe.
Much more research is needed before we can begin to dream of a reliable germ gene therapy. But somatic therapy – whose DNA changes are not inherited – is another story. Montoliu, a specialist in rare diseases , affirms that there are millions of Spanish patients suffering from this type of congenital ailments who have placed their hopes on CRISPR. The interventions carried out on their genomes would not be passed on to their descendants, they would be within legal parameters and could improve the quality of life of those affected.
Improving the efficacy and safety of these treatments should therefore be the priority. With more and more scientists dedicated to finding new CRISPR systems – using proteins that do not impose the limitations of Cas9 -, or tuning the system we already know, surely sooner rather than later we will see great advances.
Of all the existing proposals to date, the most promising have come from the same place: the Broad Institute, a joint center of Harvard University and MIT. The first, known as base editing, resembles “precision chemical surgery,” according to the director of the research, David Liu, in Nature in 2018. By using an inactivated Cas9 protein, it allows to replace isolated letters of the genetic code without cutting the DNA double helix. This is an extraordinary advantage, since the cell’s repair mechanisms are not turned on, which significantly reduces errors .
At the time, the aforementioned journal Nature predicted that “the ability to alter unique bases means that researchers can now attempt to correct more than half of all human genetic diseases.” However, the illusions fell apart when it was verified that these editors also cause numerous off-target mutations.
Even so, scientific progress in this field does not seem to be holding back. On October 21, 2019, the same group of experts from the Broad Institute presented prime editing technology – quality editing – which promises, according to its authors, the possibility of repairing 89% of the 75,000 human genetic variants associated with diseases inheritable.
The prime editing system makes use of a special Cas9 protein, altered in the laboratory, capable of cutting a single of the strands of the DNA double helix. With this premium cutter, the team led by Liu conducted 175 experiments on human cells, and succeeded in correcting the genetic causes of disorders as diverse as cystic fibrosis, sickle cell anemia and Tay-Sachs disease. “If CRISPR / Cas9 works like molecular scissors and basic editors like pencils, then we can think of quality editors as word processors,” Liu explained at a press conference.
While CRISPR is based on an enzyme capable of cutting DNA and a piece of guide RNA that defines where to intervene, this new technology works in a more complex way. It combines the cutting activity of Cas9 with another protein, called reverse transcriptase, which converts RNA to DNA. In addition, the RNA guide is also slightly different, as it not only includes the information necessary for cutting, but also contains the instructions for performing the gene editing itself.
When it finds the site where it should cut, the enzyme of this system acts only on one of the DNA strands. In that gap, the reverse transcriptase will add, letter by letter, the desired edition. Once this process is finished, the cellular machinery removes the original sequence, and the editing then goes on to integrate the genetic material, which would avoid many of the mutations that are detected when CRISPR / Cas9 is used. At the moment, the biggest problem is essentially size: the required molecular machinery is considerably more voluminous and complex than that involved in CRISPR / Cas9 and is therefore more difficult to get inside cells.
“More research is needed on a wide variety of cell types and organisms to better understand and refine prime editing ,” Liu’s team acknowledged in Nature. Montoliu, in statements to El País , was also quite cautious, and explained that other groups of scientists must now put the new tool to the test. Only that “will tell us if this innovative procedure for editing genomes is going to have possibilities and a therapeutic path or if it is going to remain as one of the dozens of proposals with alternative variants of CRISPR that we know every week,” the expert pointed out. Time will tell.
