CRISPR is an abbreviation for Clustered Regularly Interspaced Short Polindromic Repeats. This technique was introduced to the scientific world in recent years, has aroused a huge impact in the media after redesign the human embryo via this method. In 2015, he was selected by the famous Science magazine as the discovery of the year. Even though he was a candidate in 2012 and 2013, and it was not worthy of this award.
The CRISPR technique is becoming an indispensable technique in the scientific community as it allows scientists to rearrange the genome in a very sensitive, efficient and desirable manner. In recent years, with the permission of the Chinese government, Chinese scientists have announced that they have succeeded in redesigning human embryos with the CRISPR method.
“The most interesting aspect of this technique is that this method is not an invention of scientists. “
Method used by bacteria
In 1987, before the CRIPSR known, Yashizumi Ishino from Osaka University, identified the CRISPR sequences in the genome of the E. coli bacterium by coincidence. In his research on the gene IAP, he came across some repeating sequence around the gene, and in his resarch (see Figure 1) Ishino talked about this repeated seqeunces. Interestingly, there was no clue about its function for the next 20 years following this discovery.
For many years these repeat seqeunces continued to preserve the mystery. In 2002, Dutch researcher Ruud Jansen found the Cas genes in the repeated sequence. After years of work, Eugene Koonin put forward the thesis that these repeats could play a role in the defense mechanism of bacteria against germs.
Finfing Bacteriophage DNA
In 2005, three different bioinformatics studies also showed phage DNA and plasmid DNA of approximately 30-40 bases in CRISPR repeat strings. Although the presence of these non-bacterial DNAs suggests to mind the hypothesis that the CRISPR / cas system may play a role in acquired immunity and may somehow use it as a defense mechanism, the researchers’ findings were not worthy of publication by some well-known journals.
Although, not like eukaryotes but bacteria can become ill. This event is a big problem for the dairy and yogurt industry. Streptococcus thermophilus bacteria, are the cornerstones of yogurt and cheese industry. The bacteria convert lactose, a sugar type of milk, into lactic acid and are therefore used in the production of yogurt and cheese. Some types of viruses (bacteriophages) enter the bacteria and reduce their ability.
In 2007, Rodolphe Barangou from a food company turned his attention to the CRIPSR possible immune system function against viruses. Subseqeuntly, he observed that some bacteria still survive after being infected with two different types of virus, the bacteria fermenting Streptococcus thermophilus. Bacteria that were resistant to the virus showed resistance to the same virus species in the following generations. This was also a sign of some genetic change being passed on to subsequent generations.
As a result of the research, the researchers obtained more resistant bacteria for its products. At the same time, this research has shown that bacteria have a kind of immune system, thus protecting them from the effects of the same type of bacteriophage. After this breakthrough, the immune system of the bacteria suddenly became the focus of many researchers beyond the food industry.
Biochemical identification of CRISPR-Cas9
CRISPR was not only a bacterial defense, but promising many for the future. Biochemists Jennifer Doudna and Emmanuelle Charpentier, who later joined the team at the University of California, discovered the potential of CRISPR. In 2012, the researchers redesigned CRISPR and placed it into bacteria and showed that they could cut any region of the bacterial DNA with this method. A year later, they improved their work and succeeded in substituting a DNA region of their choice in the human cell lines.
Known for her research on RNA, Doudna thought that the CRISPR / Cas9 system could exert its effect through RNA. And, as she predicted, RNA has played a crucial role in this mechanism. The group, which concentrated its work on the Cas9 protein, accelerated its work on how this protein interacts with RNA and DNA and cuts double-stranded DNA. After the group had details of the mechanism of the Cas9 protein and the guideline RNA (sgRNA- single guided RNA), they began to consider how this could be modified. Their experiments have shown that this RNA sequence can be modified as desired and can bind to and cleave to the DNA to be ligated to these strands.
In the years that followed, more than 1000 studies were published, proving that this technique could be used in almost all fields and in many different species.
CRISPR and Bacterial Immunity
When bacteriophage or plasmids invade bacteria, their DNA is integrated into the CRIPSR region in the DNA of the invaded bacterium, thereby developing a defense mechanism against the next kind of invasion.
The Cas genes associated with CRISPR can encode polymerase, nuclease, and helicases, different Cas genes are introduced into the CRISPR region by simply breaking down the DNA of the phages that are invading them to a length of about 30 bases. Different Cas proteins can recognize and eliminate the phages that invade themselves, starting from these stored DNAs of phages.
Why this method important?
The Cas9 protein is easily modifiable. It functions quickly and effectively canning the entire genome. Target DNA binds to the PAM motif in Cas9 complex. The interaction of PAM with the target DNA results in the cleavage of the DNA helix and triggers Cas9 catalytic activity.
This feature of the CRISPR / Cas9 system, which allows the ability to cut and add DNA to the desired point, has excited researchers to turn this bacterial defense mechanism into a method.
Resercher were using genetic engineering even before the discovery of DNA. With selective mating methods, they allowed many animals to be viewed as they wanted. Researchers examined the possible effects of the seeds and insect eggs by exposing them to the X-ray. Thus, they have access to cereals and red large grape species used to make drinks.
Before CRISPR Zinc-finger nuclease and TALENs are the previously preferred gene editing methods, but these old techniques were both time-consuming and extremely costly. Because of these features of CRISPR method became widespread and hope for many new treatments.
CRIPR and organ transplantation
Every day hundreds of people die while waiting for organ transplants. CRISPR may be a hope in organ transplantation. For many years, researchers have been working on tissue and organ transplantation from different types of animals (Xenotransplantation) . Especially from pigs, xenotransplantation is in the forefront because the shape and size of the organs are similar to that of human beings.
However, this type of animal to human tissue transplantation can bring many problems. There are defense barriers in humans that can protect itself from external influences and interrupt the relationship with the outside environment; like our skin and our digestive system. These foreign organs cary viruses. However, many of the viruses that are harmless to other animals but harmful to us. Therefore, Xenotransplantation mediate lethal viruses pass through our defense barriers and easily spread to all our systems and cause deadly consequences.
Likewise, a virus that is harmless to monkeys can have lethal consequences in humans. The same problem exists in pigs that are genetically known to resemble humans. For example, the outbreak of swine flu in recent years has cost many people lives. Paramixovirus has also been detected in Australian swine breeders, again causing flu-like symptoms. In 1998, millions of pigs carrying the virus were culled because the Nipah virus could be fatal to humans.
To overcome this problem, George Church decided to use the CRISPR technique. Thanks to this technique, it was able to neutralize 62 porcine endogenous retroviruses (PERVs) that were not eliminated in their DNA in all pigs. Additionally, the resarchers was able to modify the 20 genes found on porcine cells that caused severe immune responses in humans after organ transplantation.
Treatment with CRISPR
The CRISPR technique can easily restore a mutated DNA sequence and can be used in the treatment of many diseases. The researchers began to reverse the mutations, which are the cause of many diseases, in the experimental environment in about 4 years.