Team:Bordeaux/HP/Silver
It is what comes to mind when we hear about CRISPR-Cas9. But what it is really hiding behind this revolutionary technique? CRISPR-Cas9 is a technique which has revolutionized the world of biology these last months. It all started in 2012 when Feng ZHANG of Broad Institute of MIT and Harvard University and Jennifer DOUDNA in collaboration with Emmanuelle CHARPENTIER of the University of California developed this system leading to a new era. This unique method offers an array of possibilities that goes beyond the dreams of both the most internationally renowned scientists that lead them to dream and non-scientists. Consequently, applications are only limited by our imagination. In research studies, it allows the precise study of a gene and its functions. If a gene is responsible for a disease, it is possible to knock it out or repair it. On the other hand, despite our knowledge on this system, we do not have any hindsight. The system is not perfect. It might be possible to cure a person with a given disease, but their child will still have that disease. For the cure to be transmissible to future generations, one has to work with stem cells or germinative cells. This equals modifying the baby in an irreversible way. In less than three decades, an uncountable number of researchers have contributed to the creation of this complex system called CRISPR-Cas9, which now leads to numerous possibilities. On 18th April 2015, Chinese scientists from GuangZhou published an article in Protein&Cell [2]. They used CRISPR-Cas9 in order to make genetic modifications on human embryos. This article was first rejected by Science and Nature for ethical reasons. These ethical problems might just stop advances in research. Since February 2016, English scientists have received permission to genetically modify human embryos using CRISPR-Cas9. [3] Our system uses a dead Cas9 which cannot cut DNA. Indeed, its two catalytic sites are inactive. We aspired to fuse this dead Cas9 to a methylase which can add a methyl group onto the DNA. This DNA methylation allows gene regulation. Moreover, it is transmissible to future generations. With this technique, DNA is not directly modified, but the expression of the target gene is: this is the principle of epigenetic. This technique modifies the expression, but not the coding sequence. As our epiCRISPR construct is transmissible to generations, it needs to be used cautiously. Indeed even if the nucleotidic sequence is not directly modified, gene expression is. Moreover this system calls for taking a step back because of its youth. Thus, in an ethical point of view our project can be dangerous. Issues which could occurs would be : a misappropriation of our system could lead to deregulations of sleep genes, superhuman which wouldn’t need to sleep, drugs which get people to sleep misused by criminals for example... Every scenario is imaginable.
Eternal youth? End of diseases on Earth? Designing babies on demand?
What is CRISPR-Cas9?
Originally, this system was a bacterial defence mechanism against virus attacks. Viruses attack bacteria by injecting their own genome into the bacterial one. Most of the time, bacteria are too weak to resist against these attacks, except when they possess a very efficient anti-viral system called CRISPR. In this case, the virus's genome is kept by the bacterial genome and bacteria keep a part of the foreign DNA in the CRISPR locus of its own genome. If the virus should attack the bacteria again, the CRISPR locus is transcribed, creating the pre-crRNA which will be cleaved within the repeated sequences and free several crRNAs. Those crRNAs hybridize with tracrRNAs which recruits the Cas9 nuclease. The guide RNA recognizes its complementary sequence, as well as the PAM pattern in the foreign DNA. This recognition induces cleavage of the DNA, and repair mechanisms fill in the gaps left by this cleavage.
The PAM pattern is at the 3’end of the virus genome. Without this PAM sequence, there cannot be cleavage of the DNA. This system keeps the Cas9 nuclease from cleaving the CRISPR locus. In order to use this system in other organisms than bacteria, scientists had the idea to combine the crRNA and the tracrRNA into a single RNA called single guide RNA. This enables the design of a sgRNA that targets the gene you want. Once the system is on the DNA helix, the Cas9 nuclease performs a double strand cut. It leads to a non-homologous end joining and by consequent a gene knocked out due to a change of the frame shift. It is also possible to insert a DNA fragment with a homologous recombination. The PAM pattern is a component of invading viruses, but it does not exist in the CRISPR locus. The sequence from the foreign DNA chosen to be inserted in the genome is always inserted in CRISPR locus. The revolution began when scientists discovered how to design a single guide RNA in order to target a specific position in the genome. The Cas9 is an endonuclease that moves on the DNA and can “jump” from one DNA to another DNA. The potential applications of CRISPR-Cas9
CRISPR-Cas9 may very well eradicate all viruses that insert their coding sequences into the genome. In our way to fight cancers, it could become a powerful tool that modifies the immune system in order to fight tumours. Many studies are currently in progress. A study [1] published in March 2016 in Nature shows that CRISPR was used to delete the coding gene of HIV in cells. In the study, almost all every cell of the rat population used had been infected by HIV. After an injection of CRISPR-Cas9 in the tail only, half of the rat cells became healthy. This technique may also work for other genetic diseases. Other advantages of the CRISPR-Cas9 system include a quicker, cheaper and more precise design compared to other existing methods.The limits of CRISPR-Cas9
The main purpose would be to cure severe human diseases rather than common ones, or physical defaults. But what will happen when modifying babies in order to cure them becomes common? Can we trust this amazing technology will not be used to modify babies to make them taller, stronger, or smarter? Being able to modify babies as we please could lead to eugenics. A break in research for ethical reasons?
As it was said during the International Summit on human gene editing in December 2015, we need to stop and ask ourselves the right questions about this technique and what it will lead to. It is very important to ask ourselves these ethical questions now, during the early stages of this technique's development, before it brings up too many important issues which could force us to step back.What about EpiCRISPR?
The advantages of our system are that methylations are precisely added, and modifications of genetic expression are easily reversible. Therefore, we do not modify the coding sequence of DNA. Our system is modular due to the customisable sgRNA. That’s why our EpiCRISPR project and therefore epigenetic techniques in general have to be used with reason and measure by scientists aware of its ethical issues and respecting the legislations of our society.
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