Introduction
Scientists have discovered a way of turning off genes so as to provide room for studying how cells work and other aspects of the cells. The discovery has been deemed to be a major success towards a news technology in studying the biochemical pathways and interactions that occur in cells hence facilitating normal development as well as disease progression. Norris (Para. 1) points out that, scientists at UC San Francisco have made this major breakthrough of turning off genes in cells. They indicate that the ability to turn off genes will pave way to increase in research and making of discoveries in biotech. Eventually, it might be possible to reprogram cells as well as regenerate tissues and organs. In so doing, it might also be a major breakthrough towards discovering the causes of cancer leading to the invention of a possible cure.
This new technology was developed by a team of scientists at UCSF Center for Systems and Synthetic Biology and UC Berkeley researchers. The team named the technology as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference which completely different from the RNA interference that is popularly used in turning off protein production. The main idea and aim of the technology is to perturb gene expression on a genome-wide measure. It helps in locating or finding any short DNA sequence in a genome and then controlling the expression of that gene. Being able to locate and control a gene expression in a genome will enable scientists to trace patterns of gene initiation and biochemical sequence of events that occur within cells. This is specifically important for scientists since they will be able to identify key proteins that usually control these events. Consequently, the disease patterns that the pathogens use such as cancer and other related ailments may be discovered and controlled (Norris Para. 3).
CRISPR Interference versus the RNA Interference
The CRISPR interference is said to allow unlimited number of individual genes to be silenced or controlled at the same time. This is contrary to RNA interference which allows a few proteins to be controlled. CRISPR technology is also accurate in the sense that only the targeted genes can be silenced contrary to FRNA interference. With this technology, the untargeted genes cannot be turned off which improves its ability to silence only the unwanted genes from a cell. Therefore, if it is possible to locate the DNA sequence that causes a disease such as cancer, there is then a very high possibility of turning off these genes and hence controlling the growth of the cancerous cells. Thus, the treatment for cancer may be found based on this technology (Norris Para. 5).
It is noted that RNA interference blocks the messenger RNA that determines protein protection centered on the blueprint confined within a genes’ DNA sequence. This is an achievement which may get rid of the problem of difficult-to-target proteins noted majorly in drug development. However, the RNA message has already been transcribed from DNA thus RNA interference does not completely solve the problem. On the other hand, CRISPR interference technology prevents the message to be written from the DNA and carrying it along hence more effective in silencing targeted genes than RNA interference (Norris Para. 6).
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
CRISPR are loci that have short direct multiple repeats present in genomes of about 40% sequenced bacteria as well as 90% sequenced archaea. It is indicated that CRISPR works in a similar manner with the prokaryotic immune system in the sense that it confers resistance to exogenous genetic features such as phages and plasmids. In essence, CRISPR technology gives a form of immunity. Short parts of foreign DNA known as spacers, are merged into the genome flanked by CRISPR repeats, and help as memory of previous exposures. Similar to RNA interference mechanism in eukaryotic organisms, the CRISPR spacers are at that point used to identify and silence exogenous genetic elements (Marraffini and Sontheime 189).
CRISPR Mechanism
Exogenous DNA is processed into small elements by some of the CRISPR-associated (cas) genes that are then inserted into the CRISPR locus. RNAs from the CRISPR loci are then articulated constitutively and managed by Cas proteins to small RNAs consisting distinct exogenously unoriginal sequence elements that have some flanking repeat sequence. These RNAs then lead or guide the rest of the cas proteins to silence exogenous genetic elements at the DNA or the RNA level. This procedure can effectively be applied in the study of normal cell interactions and that of disease development (Marraffini and Sontheime 189).
The CRISPR mechanism is known to be advantageous over other methods such as gene knockdown approach since the gene perturbation produced is reversible. Qi et al. (1175) denotes that CRISPR could be induced in an organism and reversed. This was achieved in an experiment that included the encoding of dCas9 and mRFP-specific sgRNA (NT1) together with a control of aTc-inducible promoter. By the use of inducers, cell culture responded to it quickly and there was a fluorescence reporter protein signal that showed the results of the experiment. The rate fluorescence signal that was received in this experiment was noted to be limited by the protein dilution as a result of cell growth. It was possible to repress all cells back to their normal level after a specific time. In essence, this experiment can show clearly that the silencing effects of dCas9-sgRNA can be reversed after induction (Qi et al 1175).
Factors That Determine the Efficiency of CRISPR Silencing
In addition, it is indicated that the specificity of the CRISPR must have at least a 12 bp sgRNA-DNA stretch as well as a PAM of 2 bp. This space is sufficiently big enough to cover the majority of the bacterial genomes for distinct target sites. Lastly, a study was conducted to find out whether CRISPR could be enhanced by employing the use of more than one sgRNAs that are all targeted on the same gene. It was found out that, there were distinct combinatorial effects as well as multiplicative effects. In addition, suppressive combinatorial effects were also observed by the use of sgRNAs that made their targets to overlap (Qi et al 1176). It was concluded that these effects were as a result of competition that occurred as the sgRNAs were both binding to a similar region.
CRISPR is able to Knockdown the Targeted Gene Expression in Human Cells
Research has also indicated that dependent on both thhe sgRNAs and dCas9 protein used, knock down of gene expression is possible. Repression is highly dependent on the dCas9-sgRNAs complex together with RNA-guided targeting. It was observed that in accordance with the bacterial system, it is only sgRNAs targeted to the non-anticipated strand displayed repression. Nonetheless, the regulatory effects that are found in mammalian cells showed to be dependent on the targeting locus. Thus, factors such as the local chromatin and the transcription start may be vital in the determination of repression efficiency. In essence, it is clearly indicated that the knock down of the targeted gene expressions in human beings is possible. There are only a few adjustments or improvements that are required to be made such as optimization of nuclear localization, interaction, dCas9 and sgRNA and stability (Qi et al 1177).
The fact that silencing of genes in bacteria is efficient and possible shows that it can be done in human beings. Also, the silencing is said to be very specific which indicates that there is no detectable off-target effects. Additionally, the knockdown efficiency can be achieved through changing the target loci and also the degree of the base pairing between the sgRNA and the target gene. This technology is said to be the first one to employ the use of targeted protein-RNA complex that is able to block transcription elongation inside protein coding regions. The technology is better than the RNA-based silencing which causes the destruction of the existing transcribed mRNAs.
Implications for Regenerative Medicine
The technology of CRISPR is a system used by the bacteria in defiance against viruses. It is a technology that can be used in medicine to regenerate cells in mammals without the exception of human beings. The aim is to make the dCas9 chassis to a form of an enzyme that has the ability of turning genes off and on. In that perspective, it will be possible to reprogram cells in tissues and organs for regenerative medicine. Additionally, this technology can also be used to reprogram immune cells so that they can be used in treating cancer. The success of the CRISPR technology will enable researchers to reprogram cells to be able to do things as they are instructed (Norris Para. 12).
According to the experiments conducted by researchers as noted by Cell Host & Microbe (Para. 1), CRISPR immunity prevents the natural alteration of genes both in vivo and in vitro. It is through horizontal gene transfer that takes place due to the transfer of virulence as well as antimicrobial resistance that large bacterial strains emerge. However, the use of CRISPR interference technology can help in sequencing a programmable defense mechanism that can lead to formation of a barrier to horizontal gene transfer. This too is noted to be a success in the field of medicine and in the search for a cure for cancer.
Sample (Para. 2), points out that there is a major effort that is put towards the discovery of the development of cancer cells. it indicates that CRISPR interference technology can be used by scientists to study and find out how the cancerous cells emerge. This leads to uncontrolled cell division in the body resulting to overgrowths that cause cancer. If these the causes of these abnormal multiplication and growth of cancerous cells can be established and be controlled by the use of CRISPR interference technology, scientist will be able to piece together the necessary information required to effectively produce a treatment or cure for cancer. According to the findings so far, it was indicated that there were generally a high number of ‘blackspots’ associated with cancer. It was explained that these are the regions where undergoes lots of mutations which gather at one point. By the use CRISPR interference technology, scientists are optimistic that they will be able to control mutations of the genomes hence prevent the occurrence of cancer or as a treatment mechanism.
Conclusion
In summary, the discovery of how to turn off genes so as to pave way for the study of cells is a very important step towards getting a treatment for cancer. It has been indicated that this is a major step made by scientists and researchers towards the discovery of the cure for cancer and other related diseases due to the efficiency of the approach. Experiments that have been conducted in this area using the CRISPR interference technology have shown that it is possible to turn off targeted genes only in a cell and reprogram them to do as instructed. Therefore, the cancerous genes can be studied and be targeted and turned off so that the overgrowth of cells does not occur. This is a discovery that majority of scientists are working on in order to find a total breakthrough.
Works Cited:
Cell Host & Microbe. “CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection.” Science Direct 12.2 (2012): 177-186. Web. 29 April. 2013.
Marraffini, Luciano A. and Erik J. Sontheime. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Reviews Genetics, 11 (2010): 181-190. Web. 29 April. 2013.
Norris, Jeffrey. Scientists Find More Precise Way to Turn Off Genes, a Major Goal of Treatments That Target Cancer. sciencedaily.com. ScienceDaily LLC, 7 Mar. 2013. Web. 29 April. 2013.
Qi, Lei S. et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152 (2013): 1173–1183.
Sample, Ian. Cancer scientists hope genetic markers will reveal how disease develops. guardian.co.uk. Guardian News and Media Limited, 27 Mar. 2013. Web. 29 April. 2013.
