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| CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence |
The first successful attempts to modify cellular DNA were made as early as the 1980s. After these first experiments, synthetic nucleases (proteins capable of cutting DNA) developed in the early 2000s led to many advances in genetic engineering. However, it was not until 2012 and the invention of the CRISPR-Cas9 technique by Emmanuelle Charpentier and Jennifer Doudna that genomic publishing experienced a real revolution.
Because this DNA editing tool sometimes called "DNA scissors" is indeed a revolution. Targeted, efficient and very inexpensive, CRISPR-Cas9 has been used since 2012 by many scientists from around the world in a wide range of fields ranging from oncology to bioremediation, agronomy and paleontology. But for what? Four technical specialists gathered at the Academy of Pharmacy answered this question on February 5, 2020.
Create new search models
CRISPR-Cas9 is a molecular complex composed of a so-called "guide RNA" RNA complementary to the portion of DNA that is intended to be modified and the Protein Cas9, an endonuclease capable of cutting DNA.
This complex allows targeted modifications to the genetic material of cells to be cut or made in two stages. First, the complex scans the double DNA helix and stops in front of the sequence complementary to that of the guide RNA. There, the Cas9 protein cuts the DNA. Once cut, the DNA sequence of interest can be modified if necessary, but not by the complex itself. Two systems for repairing the genetic material naturally present in the cell are then recruited and take over the complex. The first is the NHEJ system (for Non-Homologous End Joining),very effective but not accurate, mainly used to inactivate genes. The second is the HDR system (for Homology Directed Repair),more accurate but less efficient.
This CRISPR-Cas9 technique is effective on many types of organisms and cells
For example, we can create animal models. It is often a matter of modifying the genetic material of certain rodent cells so that they express a genetic disease that one wishes to study. To create these models, it is possible to collect cells from the animal, modify them by CRISPR and then re-implant them before observing the effect of the genomic modification made. Some approaches also involve modifying animal cells in the early embryonic stages so that all adult cells express the genetic modification made by CRISPR. For example, researchers have modified the genetic material of rats to contract rare diseases such as Duchesne myopathy in order to study this pathology in the laboratory, giovannangeli says.
"It is also possible to modify human cells in vitro,"she says. Changing the end of two chromosomes with CRISPR in bone cells has thus led to a better understanding of the genesis of a particular type of bone cancer: Ewing's sarcoma. "Themethod can also be used on organoids, a kind of miniature organ,"adds Michel Wassef, scientific director of the CRISPR genetic screening centre at the Institut Curie in Paris, who was also invited to speak by the Academy.
Finally, all RNA guides to the research complexes can be centralized and stored in vast libraries that can accelerate discoveries. Today, there are libraries of RNA guides murin or human such as the genetic screening platform CRISPR led precisely by Michel Wassef.
Treating cancer or genetic diseases but not only
"In therapeutics, we try to use CRISPR-Cas9 to add, correct or inactivate certain genes involved in the development of diseases," explainsCarine Giovannangeli. This tool could therefore find applications in the treatment of all diseases caused either by the presence of a defective gene (genetic diseases) or by the mutation of a gene (cancers). "Todo this, two approaches are being considered,"says the researcher. The first is called ex vivo: it involves taking a patient's cells, cultivating them and modifying their genomes using IN vitroCRISPR, and then re-implanting the modified cells into the patient. The second approach is called in vivo: it is to modify the genetic material of the patient's cells directly into the body. The latter approach seems particularly difficult to implement.
Regarding the management of genetic diseases linked to a defective gene, the main proofs of concept have been made for sickle cell disease and beta-thalassemia, which cause severe anaemia. In both pathologies, a gene is responsible for an abnormality of hemoglobin then unable to properly store oxygen within red blood cells, also called hematias. Attempts to reactivate a gene encoding another type of hemoglobin, known as fetal hemoglobin, have been made to replace hematias containing abnormal hemoglobin with hematias containing functional fetal hemoglobin.
In oncology, research and clinical trials assess the effectiveness of two major types of strategies. The first is to directly target the tumor genome: researchers are either trying to activate tumor suppressor genes or inactivate so-called oncogene genes. The second strategy - the most common and currently the most effective - aims to stimulate the cells of the immune system so that they attack massively and specifically the tumor. Car-T cells are an example of immunity cells modified for this purpose. In oncology, the challenge is to be able to offer patients highly personalized treatments that would maximize the effectiveness of management while reducing their side effects.
But DNA scissors also find applications in infectious diseases. First, in virology, fairly advanced clinical trials show that CRISPR could be used to fight HIV. Studies focus in particular on modifying a gene present in human cells encoding a virus receptor: it involves inactivating this gene in order to rid the cells of this receptor and prevent the virus from entering it, says Carine Giovannangeli. Less famous experiments have also been carried out in bacteriology with the aim of combating antibiotic resistance. "Theaim is to introduce RNA-protein complexes into bacteria in order to inactivate their antibiotic resistance genes," explainsPhilippe Glaser, head of the Ecology and Evolution of Antibiotic Resistance Unit at the Pasteur Institute in Paris and the third researcher to speak at the Academy of Pharmacy.
And the CRISPR method could also be used in many other fields such as ophthalmology, cardiology, parasitology, etc. A total of 30 clinical trials are currently underway to test therapeutic applications of extremely varied DNA scissors.
Creating new foods and materials or studying the genomes of extinct species
In fact, CRISPR-Cas9 can be used in all areas where living cells are used.
For example, this tool can therefore be used in synthetic biology, a discipline that aims to create materials: DNA scissors could be used to produce new biofuels using microorganisms with modified genomes, says Alain Puisieux, director of the research center of the Institut Curie also present on February 5. "Inthe field of bioremediation, the CRISPR-Cas9 tool is studied on bacteria so that they can be equipped with an optimized metabolism capable of degrading plastics,"he adds. Paleontologists would also use CRISPR to study the genomes of extinct species such as the mammoth.
But the other than therapeutic applications that are of greatest interest and concern are agronomy and ecology. This technology makes humans able to continue to easily and cheaply modify the genomes of plant crops and livestock. For example, different teams would work on the development of gluten-free wheat varieties. "CRISPRisés" foods have already been approved by the FDA and are subject to appeals for marketing approvals in the European Union, Giovannangeli says.
Some limitations of the technique: towards new genome-editing technologies?
"The limits of the CRISPR technique are twofold,"says Alain Puisieux. They are primarily technological because CRISPR still lacks efficiency and specificity. An important limitation concerns so-called "off-target" effects of the technique, which sometimes leads to unforeseen genome cuts at different sequences of the target sequence. However, this difficulty may be being circumvented by the emergence of new genomic editing techniques derived from CRISPR: base editing and prime editing.
But the limit, which according to all specialists will be the most difficult to overcome, is ethical. For example, experiments disapproved by the international community that led to the birth of babies with the genome modified by CRISPR took place in 2019. The marketing of modified foods also raises many questions. Should all research based on the use of CRISPR be banned? "Whatis certain is that the key element that will ensure ethical science lies in education and the ability of everyone to look at their own practices with hindsight,"concludes Alain Puisieux.