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There are numerous pressing concerns in the field of medicine about Clustered Regularly Interspaced Short Palindromic Repeats (CRISP), which is a great gene editing technique (Lundgren, Charpentier, & Fineran 2015). It has now become one the biggest game changers that have ever hit biology since Polymerase Chain Reaction (PCR). CRISPR, which is present in almost all microorganisms such as archaea and bacteria, normally consists of short repetitions with each of the repetition being followed by some spacer DNA hence making it possible for the researchers to now change and alter the DNA of almost every organism.
Brief History
The first scientist to discover CRISPR was Ishino and his colleagues in 1985 and found out the existence of fourteen repeats each composed of 29 bp that were linked by DNA spacer. Later, analysis, which was conducted by Mojiva further revealed that CRISPR is found nearly in ninety percent of the archaeal genomes and forty percent of the bacteria genomes Sharma 2016). More contributions to the discovery of CRISPR were made when Jansen identified the presence of unique genes known as CRISPR Associated Sequence (CAS), which border the array of CRISPR.
Structural Features
According to researchers such as Conklin, the main components of CRISPR system are CRISPR array and CAS genes. The majority of the existing genomes only have one locus of CRISPR. There is more diversity among species on repeats, which are 24-47 in length. Spacers can be derived from both sense, and the antisense strand in each array they are found in where they are evenly distributed in the genome of a phage (Futuyma, Shaffer & Simberloff 2016). The six types of Cas genes which include Cas 1-6 belong to the large family of proteins which have a high association with the CRISPR system. Exonuclease, helicase, endonuclease, RNA binding, DNA binding and the transcription regulator are the main functional domains of the Cas proteins.
Function
The CRISPR molecule plays an important role, especially in the defense mechanisms of living organisms against phage infections. According to the discovery made by a biologist known as Barrage, when the transformation of the sets of the repeat-spacer sequence into phage sensitive bacteria occurs, a strong resistance to phage infection takes place in the bodies of the new mutants (Peng 2015). That becomes possible through the adaptation abilities of the phages to the existing situation by changing the PAM sequence, which helps the organism to withstand resistance against bacteria.
Mechanism
Scientists claim that adaptation, expression, and interference are the three stages of CRISPR defense mechanism. In the adaptation stage, full recognition of the new spacer from an external genetic source takes place. That is then followed by the processing of the proto-spacers sequences into smaller spacers, which then become integrated downstream into the CRISPR array (Wu 2014).
The transcription of the CRISPR array into a very long mRNA transcript marks the start of the biogenesis or expression stage. The cutting of the middle of the repeats by mRNA results into short RNA which comprises of two half repeats and one spacer in both ends. A complex functional Cas protein is then formed after the binding of the short RNA with Cas protein takes place.
Researchers argue that the mechanism process ends once there are recognition and binding of the Cas protein with the different sequence as its target takes place. This action results in degradation of the compound Cas protein with an activity similar to the RecB.
Classification
Scientists have classified the CRISPR system into three types. The type I CRISPR consists of six different subtypes, which include I-A – I-F, and it is most common in bacteria and archaea. The C-terminal DExH helicase and N-terminal HD phosphohydrolase domains are the components of the Cas-3 gene on which these six subtypes encode. These subtypes usually work together to unwind and cleave the target dsDNA. However, due to the inability of the Cas-3 to recognize the target DNA, it is essential for other proteins complex to be present in this system. Cascade, which is a protein complex in each of the subtypes of the type I CRISPR acts as a surveillance complex and assists in recognizing the presence of foreign DNA in a living organism. The sophistic organization of the Cascade complex enables it to protect the crRNA from degradation efficiently and ensures that it is continuously able to pair with the target sequence.
Type II CRISPR-Associated System is the second type of the CRISPR, and it is composed of four Cas genes which include Cas 1, Cas 2, Cas 9 and csn2 (II-A) or Cas 4 (II-B). This type of CRISPR is only found in bacteria, and the involvement of a new kind of RNA called tracrRNA (trans-encoded RNA) makes this system unique. Its function mainly involves recognizing the repeat sequence in the pre-crRNA and then automatically directing the RNase III, which houses it to cleave the target spacer-repeat quickly (Futuyma, Shaffer & Simberloff 2016).
The last type of the CRISPR is the type III CRISPR-Associated System that is commonly found in archaea and is subcategorized into III-A which frequently targets the DNA and III-B which targets the RNA, and these two subtypes encode the Cas6 and Cas10. Soon after encoding, the Cas6 functions like endoribonuclease by cleaving the pre-crRNA into crRNA.
Application
The majority of the geneticists claim that CRISPR, which is a gene-editing method in the field of medicine is the cheapest, easiest and quickest to use. Today, life sciences are greatly being influenced by CRISPR as elaborated below.
We can do it today.
Editing Out Diseases
The crucial step to use the CRISPR technology for gene therapy in human beings took place last year when a bioengineer, Daniel Anderson with the help from his colleagues corrected tyrosinemia, a human mutational metabolic disease through using CRISPR to study mice. Today, the primary reason for happiness in the biotechnology and scientific field is the already confirmed possibility of this gene-editing tool to accelerate the field of gene therapy. Anderson claims that the remarkable attempts by scientists and researchers to discover gene therapy based on CRISPR will see to it that clinical experiments aimed at finding the cure come into existence before the end of the following year. That will see to it the possibility of the injection of the CRISPR components directly into body tissues of a living organism such those in the eyes (Eaglesham & Hardy 2015). Moreover, there will be the chances of removing the cells from the body of the organism, engineering them in the lab and then finally putting them back in the body. In this case, human diseases will be eliminated. For instance, there will be the possibility correcting the blood-stemming cells to cure and prevent thalassemia among other ailments. Researchers are optimistic that the CRISPR is going to help in eradicating the present diseases associated with genetics in a broader perspective.
CRISPR on the Farm
Right before the discovery of CRISPR, biologists and researchers have been using expensive gene-editing tools, which involve random insertion of a gene into the genome, which is then followed by sequences coming from bacteria and viruses, which influence the response. These methods have always been inefficient and are highly criticized by farmers who worry that this kind of insertion can interrupt other genes present in the plants. Besides, it is tough and expensive to get the approval of the genetically modified crops (Eaglesham & Hardy 2015). However, this situation has started changing with the introduction of the CRISPR in the agricultural sector. It is viable for farmers to use this gene-editing tool, as it is less costly and easy to use. Over the past few months, scientists and researchers have been using this method to make rice and wheat that are disease-resistant plus engineer small pigs and other livestock. There is remarkable progress by the researchers on engineering disease-resistant goats, dehorned cattle, and oranges enriched with vitamins.
Engineered Ecosystem
Different existing articles such as, “Engineering a Revolution in Gene Editing” by Tim Wang prove that researchers have already deployed the CRIPSR on the organisms in the world. A gene drive, which can sweep an edited gene through a selected population, has been used to help in wiping out disease-carrying ticks and mosquitoes, eradicating herbicide resistance in plants such as pigweed and eliminating invasive plants, which plague farmers (Eaglesham & Hardy 2015).
Research Revolution
Biologists have long been using molecular tools, for instance, the models of genetically modified mice to edit genomes. According to a molecular biologist at the University of Brandeis in Massachusetts by the name James Haber, the adoption of these molecular tools for biological studies in the labs has over the past one decade being impossible due to the difficulty of engineering them plus their expensive costs (Eaglesham & Hardy 2015). This challenge has being solved by the discovery of CRIPSR in which the researchers only need to make an order of the RNA fragment when the need be and even buy the various ingredients needed off the shelf, which are cheaper than the other gene-editing tools used to study genetics. The continued use of the CRISPR by the researchers is going to revolutionize and contributed to significant discoveries now and in the future.
Recommendations and Conclusion
Sources show that there are safety and ethical concerns from both the public and private sectors of the United States regarding the numerous experiments that the researchers are doing using the CRISPR. There are worries that the already edited organisms may disrupt the whole ecosystems. For this reason, the government has requested the scientists concerned with the CRIPSR to conduct further studies to prove that this technique is fit for use on the living organisms and whether it generates potentially risky genome edits and strays.
- Eaglesham, A., & Hardy, R. W. 2015. New DNA-editing Approaches: Methods, Applications and Policy for Agriculture: Proceedings of the Twenty-Sixth Annual Conference of the North American Agricultural Biotechnology Council, Hosted by Cornell University and Boyce Thompson Institute, October 8-9, 2014. Ithaca, NY: North American Agricultural Biotechnology Council.
- Futuyma, D. J., Shaffer, H. B., & Simberloff, D. 2016. Annual Review of Ecology, Evolution, and Systematics. Palo Alto, CA: Annual Reviews, Inc.
- Sharma, I. 2016. Development of Whole-Genome CRISPR-Cas9 Functional Screens for the Discovery of the TH-302 Sensitivity Genes.
- Lundgren, M., Charpentier, E., & Fineran, P. C. 2015. CRISPR: Methods and Protocols. New York: Humana Press.
- Peng, W. 2015. Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus Islandicus. Copenhagen: University of Copenhagen, Faculty of Science, Department of Biology.
- Wu, X. 2014. The Mechanism and Function of Pervasive Noncoding Transcription in the Mammalian Genome.