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CRISPR/Cas9 is a new technology for editing the genomes of higher organisms based on the immune system of bacteria. The basis of this system is special sections of bacterial DNA, short palindromic cluster repeats, or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Between identical repeats, DNA fragments are distinguished from each other – spacers, many of which correspond to parts of the genomes of viruses that parasitize this bacterium.
When a virus enters a bacterial cell, it is detected using specialized Cas proteins (CRISPR-associated sequence) associated with CRISPR RNA. If a virus fragment is recorded in the CRISPR RNA spacer, Cas proteins cut viral DNA and destroy it, protecting the cell from infection. At the beginning of 2013, several groups of scientists showed that CRISPR/Cas systems could work not only in bacterial cells but also in cells of higher organisms, which means that CRISPR / Cas systems make it possible to correct incorrect gene sequences and thus treat human hereditary diseases.
Biochemistry of CRISPR/Cas9
In order to fix the problems or “wrong” gene, a very precise molecular scalpel is needed that can find the mutant nucleotide sequence and can cut it out of DNA. Cas9 is such a scalpel, and with the help of the RNA guide, the series of which coincides with the desired location, it can introduce a gap in the desired area of the genome. Recognition of the target occurs on a site with a length of 20-30 nucleotides (Katz & Pitts, 2017).
On average, sequences of this length are found in the human genome once, which allows for accuracy. A cell will not die from a DNA break since it will be corrected by a healthy copy from a paired chromosome due to the natural process of DNA repair. If there is no paired chromosome, as in the case of hemophilia, a portion of the correct gene can be added to the cell simultaneously with Cas9 and the RNA guide and use as a matrix to heal the introduced gap.
Using CRISPR/Cas9, a researcher can do multiplex editing of several incorrect genes at once. In order to do this, one must enter the Cas9 protein and several different RNA guides. Each of them will direct Cas9 to its own target, and together they will eliminate the genetic problem. In general, the described mechanism functions due to the principle of complementarity of double-stranded DNA (Chapman, Gillum, & Kiani, 2017).
DNA double helix chains recognize each other according to the rules of complementarity. CRISPR RNA identifies its targets in double-stranded DNA in the same way, thus forming an unusual structure containing a double-stranded region of mutually complementary RNAs and one of the target DNA strands, and the other DNA strand will be extruded.
Impact and Implication
First of all, with the help of CRISPR/Cas9, people will be able to treat simple, monogenic genetic diseases such as hemophilia, cystic fibrosis, and leukemia. In these cases, it is clear what needs to be edited, but there are diseases with high heritability, the genetic nature of which is very complex. Such disorders are a complex result of the interaction of different genes and their variants. For example, many scientists are looking for genes for schizophrenia and alcoholism; every year, they find new ones, every year, a part of previously discovered genes has nothing to do with it (Wade, 2015). How to treat such complex diseases with CRISPR/Cas9 is not clear, and multiplex approaches will be required.
It is important to understand that the practical application of CRISPR/Cas9 in medicine is rather a distant future, and it will take a lot of work, improving the technology, reliability, and safety. In general, the situation with blood diseases is better since the damaged gene is needed only for hematopoiesis, and cell therapy technologies of such illnesses are well developed. For instance, if a person has leukemia and in order to eliminate the disease, he or she will be irradiated, then they will find a suitable donor and transplant the bone marrow (Katz & Pitts, 2017). It’s a long time to look for a donor, but there is never a complete immunological match.
Using the CRISPR/Cas9 system, people can obtain a patient’s bone marrow sample and heal his or her hematopoietic stem cells by changing the wrong letter. Then the patient will have to be irradiated to kill the affected hematopoietic cells and introduce his or her edited cells back – not the ones of the next of kin or a stranger, but precisely his, which are fully compatible. They will begin to divide and produce healthy blood cells.
In regards to editing, for example, a liver tumor, everything is much more complicated (Chapman et al., 2017). It will be necessary to solve the main medical problem: the problem of delivering components of the CRISPR/Cas9 system to the affected cells. In 2015, Chinese scientists attempted to correct the genome of a human embryo (Katz & Pitts, 2017). They took a fertilized human egg with a spoiled gene leading to beta-thalassemia blood disease.
Cas9 protein and an RNA guide were introduced into the cell, which was supposed to find and cut the wrong copy of the gene, followed by repair using a healthy matrix. As a result of the experiment, in 5-10% of embryos, the mutation responsible for the occurrence of the disease in adults was indeed corrected (Katz & Pitts, 2017). However, the bad news was that in all cells of the treated embryos, there were a large number of mutations that did not appear at all where it was supposed to.
Thus, the technology needs to be improved because it is not accurate enough. Exact editing is obtained when a portion of the target DNA with a length of a little more than 20 nucleotides complementary interacts with a completely corresponding RNA guide (Wade, 2015). Unfortunately, a large number of variants of the target sequence can exist in the genome, differing from it by only one letter or two, and so on. Each of these variant targets interacts worse than a perfectly suitable target. However, since there are many such sequences, it is complicated to avoid incorrect recognition. Scientists need to improve the specificity of the Cas9 protein and choose guides very carefully.
In conclusion, today, CRISPR is one of the most popular technologies, and many young people, students, are dreaming about working with CRISPRs. Now, these studies are becoming generally technological, and fundamental questions are few. A section of genomic DNA at the level of a defective gene and restoration of the edited DNA region of homologous recombination by several orders of magnitude. At the same time, there is another system of DNA damage repair in the cells, called the non-homologous connection of the ends, when the integrity of the DNA is restored without completion due to the lack of a matrix.
However, the CRISPR/Cas9 editing system is currently imperfect, because as a result of its work, for several reasons, there is the possibility of incorrect PHK binding and the appearance of so-called non-targeted effects, resulting in random DNA cuts and, as a result, insertions and deletions. How to minimize the latter and increase the likelihood of homologous recombination are tasks that many laboratories are trying to solve.
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Chapman, J. E., Gillum, D., & Kiani, S. (2017). Approaches to reduce CRISPR off-target effects for safer genome editing. Applied Biosafety, 22(1), 7-13.
Katz, G., & Pitts, P. J. (2017). Implications of CRISPR-based germline engineering for cancer survivors. Therapeutic Innovation & Regulatory Science, 51(6), 672-682.
Wade, M. (2015). High-throughput silencing using the CRISPR-Cas9 system: A review of the benefits and challenges. Journal of Biomolecular Screening, 20(8), 1027-1039.