Selective mutation and DNA marking were the leading therapeutic and disease modeling intrusion technique. The application of this technique has a substantial effect on comprehending the root of the genetics behind numerous carcinomas. For instance, procedures such as miRNA intervened marking, have formulated an excellent way of diagnosing and prognosis of diseases, particularly in colon carcinomas (Varni, Limbers & Burwinkle, 2007). Nonetheless, because of costly and labor-intense practical issues of DNA engineering, those techniques are not commonly applied in the community of biomedical research. But, currently, gene therapy has been advanced with the discovery of a programmable nuclease called the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) structure.
CRISPR is the repetitive sequence of DNA. They were first seen in bacteria having spacer DNA sequences between the repeats that matched viral sequences. Later, it was noted that upon viral infection, the bacteria translate these DNA elements into RNA. The RNA directs a nuclease towards viral DNA to cut it, offering defense against the virus (Fineran & Charpentier, 2012). The nucleases are termed as Cas, which stands for CRISPR-associated. The CRISPR or Cas system is internally present in a broad range of bacteria and functions as a protection system against attacking viruses (Deshpande, Vyas, Balakrishnan & Vyas, 2014). Notably, the portion of the CRISPR serves as an immune memory storing a specific viral sequence of the DNA between segments of interspaced palindromic repeated sections.
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Consequently, the portion of the Cas segment of the system functions to cut the DNA of attacking viruses. Important to note, the connection between these two elements of the system decide the effectiveness and strength of the essential DNA editing. CRISPR transforms the specific viral DNA between its recurring sections into two RNA moieties, including tracrRNA and crRNA (Deshpande, Vyas, Balakrishnan & Vyas, 2014). These two RNA moieties are the past decisive enzymatic activity and the final Cas cell, firmly binding on to any genetic element from attacking viruses that correspond with the crRNA. Upon latching, the Cas enzymes cut the marked DNA, which prevents further replication of viruses.
The strength and accuracy of CRISPR make it a suitable instrument for biomedical and medical developments. Using a specific Cas enzyme harvested from streptococcus pyogenes (Cas9), with a lone chimeric RNA (sgRNA) guide enclosing both crRNA and tracrRNA, makes the CRISPR system mark virtually all sequence in a cell (Deshpande, Vyas, Balakrishnan & Vyas, 2014). However, for Cas9 to latch to and cut the marked substrate, the pairing of the base between the marked DNA and the sgRNA 5' end must take place. Therefore, by changing the sequence at the sgRNA 5' end, Cas9 can beneficially be programmed to mark an extensive range of series, which triggers the breaking of DNA double-strand protecting the cells from invading viruses.
Unfortunately, the extensive usage of CRISPR certainly generates apprehensions and ethical issues following its application in the biomedical research community. Despite its numerous associated benefits, modulation and editing of genes are associated with multiple concerns across the field of the biomedical community. To mention, among the issues related to the application, CRISPR includes the capability to guarantee that only the marked gene is edited and harmful variations to the health of a person are not induced across the genome. Besides, additional off-target location mutations might consistently result in further disease and unwanted phenotypes (Anderson et al., 2015). Recent research, according to Brugge and Missaghian (2006), has also achieved to formulate CRISPR gene drives that permit the next evolution and spread of an edited gene across the entire population, which conceives ethical and political issues for commercial application as well as the advancement of CRISPR.
References
Anderson, E. M., Haupt, A., Schiel, J. A., Chou, E., Machado, H. B., Strezoska, Ž., ... & van Brabant Smith, A. (2015). Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity. Journal of biotechnology , 211 , 56-65.
Brugge, D., & Missaghian, M. (2006). Protecting the Navajo people through tribal regulation of research. Science and Engineering Ethics , 12 (3), 491-507.
Deshpande, K., Vyas, A., Balakrishnan, A., & Vyas, D. (2014). Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Genetic Engineering: A Novel Therapeutic Approach in Colon Carcinomas. Am. J. Robotic Surg , 1 , 1-4.
Fineran, P. C., & Charpentier, E. (2012). Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology , 434 (2), 202-209.
Varni, J. W., Limbers, C. A., & Burwinkle, T. M. (2007). How young can children reliably and validly self-report their health-related quality of life?: An analysis of 8,591 children across age subgroups with the PedsQL™ 4.0 Generic Core Scales. Health and quality of life outcomes , 5 (1), 1.
