In 2020, the Nobel Prize in Chemistry was awarded to French microbiologist Emmanuelle Charpentier and US biochemist Jennifer A. Doudna "for their contributions to the development of theCRISPR-Cas9system, which allows cutting and reassembling DNA chains".
So, what is the CRISPR-Cas9 system, which has been used in genetic research worldwide following the discovery made by Charpentier and Doudna?
WHAT IS CRISPR-CAS9?
CRISPR-Cas9, also known as"Technology that can perform surgery on DNA", is an application that created excitement in the scientific world with the 2020 Nobel Prize in Chemistry. In its shortest and most accurate form, CRISPR-Cas9 is a genome editing tool. In other words, it is a unique technology that allows geneticists and medical researchers to add, subtract or change the sequence of DNA. Faster, cheaper and more accurate than any of the techniques used to date, CRISPR-Cas9 has a wide range of applications.
The elements that make up CRISPR-Cas9, which is the simplest, most versatile and sensitive technique currently available among genetic manipulation methods, can be analyzed as follows:
CRISPR stands for "Clustered Regularly Interspaced Palindromic Repeats". Although there is not yet a clear translation of the definition in Turkish, it is possible to translate it as "Clustered Regularly Interspaced Palindromic Repeats".
What is meant by CRISPR are the gene sequences that define the CRISPR locus on the DNA sequence of the living organism. These are the cas genes, followed by the leader sequence and then the repeat and spacer sequences. Although the repeat sequences are exactly the same for a living organism, the spacer sequences between these repeats differ from each other.
Cas is the general name of the proteins involved in this immune system.
HOW LONG HAS CRISPR-CAS9 BEEN KNOWN?
The existence of CRISPR clusters has been known since the 1980s. However, the role of CRISPR in the defense mechanism of living beings was proven quite recently, in 2005, with the research and discovery of 3 different groups working on CRISPR genes.
What was discovered was that the interval genes found in CRISPR clusters have the same sequence as some of the viruses that infect that organism. Having a gap gene with the same sequence as a virus DNA created a resistance against that virus.
As a result of the research, the hypothesis that the CRISPR system could be a nucleic acid-based immune mechanism was tested and this claim was largely confirmed.
In 2012, a team led by Jennifer Doudna developed an application that could cut, isolate and edit the DNA of a living organism. In short, all an active Cas9 protein needed to cut DNA with high precision and accuracy was a scissors that could cut the sequence of that DNA. Based on this, an RNA molecule corresponding to this base sequence of DNA was created and this RNA molecule was combined with the Cas9 protein to form a complex.
CRISPR-Cas9 technology enables cutting from the desired DNA region.
HOW DOES CRISPR-CAS9 WORK?
The CRISPR-Cas9 system consists of two important molecules that create changes (mutations) in DNA:
An enzyme called Cas9: It acts as a "molecular scissors" that can cut two strands of DNA at specific places in the genome. This allows DNA fragments to be added or removed.
A piece of RNA called guide RNA (gRNA): It consists of a small (about 20 bases) pre-designed RNA sequence inside a longer RNA skeleton. The long RNA skeleton binds to DNA and the predesigned sequence guides Cas9 to the right spot in the genome. The Cas9 enzyme then cuts in the right places.
In CRISPR-Cas9 technology, a guide RNA with RNA bases complementary to the target DNA sequence in the genome is designed to find and bind to a specific sequence in the DNA. The enzyme Cas9 follows the guide RNA to the relevant location in the DNA and creates a cut in both strands of the DNA. At this stage, the cell recognizes that the DNA has been damaged and tries to repair it. Using the DNA repair mechanism, scientists can modify one or more genes of the cell of interest.
CRISPR: A GAME-CHANGING GENETIC ENGINEERING TECHNIQUE
Described by many researchers working in the field of molecular biology and genetic engineering as "an astonishing technique", CRISPR has so far been tested in many living organisms, including human stem cells, with very positive results.
In 2013, Science magazine named the development of CRISPR "one of the most important scientific advances of the year". The 2020 Nobel Prize in Chemistry was awarded to Charpentier and Doudna "for their contribution to the development ofCRISPR-Cas9".
More work needs to be done to perfect the technique, but it is clear that successful treatments are possible in a wide range of fields.
WHICH DISEASES WILL BE TREATABLE WITH CRISPR?
So which diseases will it be possible to use CRISPR-Cas9 technology to treat?
It is possible to use CRISPR-Cas9 technology in the treatment of many medical conditions with a genetic component (cancer, hepatitis B and even high cholesterol).
IS CRISPR-CAS9 TECHNOLOGY RISKY?
CRISPR-Cas9 systems also face various challenges while working on DNA. Foremost among these is "off-target effect".
In theory, the CRISPR-Cas9 system is specific and works according to the expert's intended design, but in practice this is not always possible. Once the system is up and running, it can create mutations elsewhere in the genome, known as "off-target modifications".
Notably, off-target effects are random and can affect other genes or other regions of the genome.
ETHICAL CONTROVERSIES RELATED TO CRISPR
There are no ethical issues with the use of CRISPR-Cas9 or similar gene editing technologies on bodily cells.
To date, CRISPR-Cas9 technology has been used in a small number of cases to treat fatal conditions. However, the possibility of using CRISPR-Cas9 or similar gene editing technologies to edit reproductive cells raises many ethical questions. Since the changes to be made on reproductive cells ( germline in English) will be passed on from generation to generation, there is a serious ethical dimension to the issue.
The CRISPR method has attracted a lot of attention in a short time and has received many awards, including the Nobel Prize, but it is not yet possible to talk about definitive results. Experiments are being conducted with the CRISPR/Cas9 technique on human stem cells, but testing the system in humans has not been realized for now due to reasons such as the unpredictability of the results of the method and the 20%-30% success rate in monkeys.
CRISPR-CAS9 TECHNOLOGY ON THE AGENDA OF ÜSKÜDAR UNIVERSITY
CRISPR-Cas9 technology, which was first studied in 2012, has been followed by Üsküdar University since it entered the agenda of the world of genetics and molecular biology.
TURKEY'S FIRST APPLIED CRISPR-CAS9 WORKSHOP
Turkey's first practical workshop on the CRISPR/Cas9 method was held at Üsküdar University in June 2016. A large number of academic staff from universities in Erzurum, Uşak, Afyon, Konya, Gebze TUBITAK-MAM, Sakarya, Izmir and Istanbul participated in the 1-day workshop, where the first session was in silico training followed by applications in the laboratory.
In 2016, the 2nd CRISPR-CAS9 DNA Surgery Workshop was hosted by Üsküdar University. Like the first one, the workshop, which was attended by a large number of academic staff, physicians, students and professionals from various universities in Turkey, especially in Istanbul, was carried out with in silico/computerized training and application in the laboratory.
ÜSKÜDAR UNIVERSITY WILL CONTRIBUTE TO NATIONAL DRUG DEVELOPMENT WITH TRGENMER
In 2018, Üsküdar University Transgenic Cell Technologies and Epigenetics Application and Research Center TRGENMER was established within Üsküdar University with the regulation published in the Official Gazette and started its activities.
TRGENMER, which aims to carry out studies on transgenic cell technologies, which play an important role in the elucidation of disease mechanisms, the development of new medical treatments based on these mechanisms and more specifically in the production of recombinant proteins, and the CRISPR-Cas9 system, which can be used in the elucidation and treatment of many diseases, especially cancer treatment in the future, will contribute to the position of our country in the world in this field and will be instrumental in the development of national drugs by investigating epigenetic mechanisms using transgenic cell technologies and developing drug candidate molecules based on these defined mechanisms.
