CRISPRtechnology in research and beyond:Abstract: In last few years a revolutionary technologyhas gained a lot of attraction in the scientific community. Researcher aroundthe globe have adopted this new technology that facilitates making specific manipulationof desired DNA of humans, animals, microbes and plant. As compared to priortechniques used for the modification of DNA, this new technology is very much easierand rapid. This technology is called as “CRISPR” (Clustered RegularlyInterspaced Short Palindromic Repeat) and this technology not only help useasier way to conduct research procedures but also it helps in curing thediseases. Clustered Regularly Interspaced Short Palindromic Repeat this nameitself states the distinctive organization of short and moderately palindromicrepeated DNA sequences found in the genome of many bacteria and other microbes.Whereas supposedly harmless,CRISPR sequences are very essential constituent of the immune system of theabove organisms.
As we know immune system responsible for the protection of theliving organism’s health. Like all other living animal, bacterial cells can be attackedby viruses. Which are small, contagious agents. If a virus causes an infectionto the bacteria, the CRISPR immune system of that bacteria gets activated andprotect it from that virus by killing it. CRISPR immune system destroys thegenomic material of that virus so that it cannot replicate itself and harm the hostcell. Only just, CRISPR related protein 9 (Cas9) has been used as an important meansin the field of biotechnology 1,2,3,4. Introduction:CRISPR gene editing is taking biomedical research by storm.
Providingthe ultimate toolbox for genetic manipulation, many new applications for thistechnology are now being investigated and established. CRISPR systems arealready delivering superior genetic models for fundamental disease research,drug screening and therapy development, rapid diagnostics, in vivo editing andcorrection of heritable conditions and now the first human CRISPR clinical trials. Thecontinuing patent battle for CRISPR-Cas9 licensing rights and the emergence ofnew editing systems such as Cpf1 has so far done nothing to slow the advance ofCRISPR-Cas9 as the leading gene editing system. There are weekly press releasesand updates on new advances and discoveries made possible with this technology;the first evidence is now emerging that CRISPR-Cas9 could provide cures formajor diseases including cancers and devastating human viruses such as HIV-1 The key to CRISPR-Cas9’s uptake is its ease of applicationand design, with retargeting only a matter of designing new guide RNA. It hasquickly surpassed TALENs (Transcription Activator-Like Effector Nucleases) andZFNs (Zinc Finger Nucleases) where editing, now possible with CRISPR, waspreviously prohibitively complex and time-consuming. As well as correcting genemutations with scar-less modifications, with CRISPR-Cas9 it is possible tocontrol the expression of entire genes offering longer term expressionalteration compared to other methods such as RNAi. CRISPR-Cas9 systems,tools and basic methodology are very accessible as ready to go toolkits thatanyone with lab space and an idea can pick up and start working with.
This isthanks largely to the efforts of Addgene andcommercial service and product providers. Alongside CRISPR research there areinnovations in companion technologies and design software. In response to agrowing need, companies such as Desktop Genetics havedeveloped open access software to accelerate CRISPR experimentation and analysis. Mechanism: Figure 1: The steps involved in CRISPRmechanism.
CRISPRs are sections in thegenome of bacteria which helps to protect against viral infection. Thesesections constitute short DNA repeats (black diamonds) and spacers (colouredboxes). When a formerly unknown virus infects bacteria, a new spacer from thevirus is merged between current spacers. The CRISPR system gets activated, ittranscribed and proceed to produce short CRISPR RNA molecules. The CRISPR RNAlinks with and guides the bacterial CRISPR machinery to an identical targetsequence in the infecting virus. The CRISPR machinery cut and destroy theinfecting virus genome. Thesespacers are derivative of virus DNA that have previous infection to the hostbacteria.
Therefore, spacers work as a ‘genetic memory’ of earlier infections.If another infection by the previous same virus occurs, then the CRISPR systemwill destroy any similar spacer sequence and will defend the bacteria fromviral infection. If a totally new virus infects the bacteria, then new spacerswill produce and incorporated in the chain of spacers. The CRISPR systemprotects bacteria from viral infection by three basic steps such as steps 3,5.
Step 1.Adaptation: DNA of infecting virus is administered into short fragments thatare inserted into CRISPR sequence as new spacers.Step 2. CRISPR RNA Production:CRISPR sequence and spacers in the bacterial DNA sequence go throughtranscription. This RNA chain is spliced into short fragments called CRISPRRNAs.Step 3.
Targeting: CRISPRRNAs guide bacterial CRISPR machinery to kill the viral genetic material.Because CRISPR RNA sequence are copied from the sequence of virus DNA duringthe process of adaption. They are precise matches of the viral genetic materialand therefore acts as excellent guides.The specificityof CRISPR immunity mechanism in identifying and killing infecting viruses isnot just beneficial for bacteria. Inventive applications of this primeval yet gracefulprotection mechanism have arisen in disciplines as various as medicine,research, and industry.Applicationsof CRISPR technology:1. In Industry:The integral functionsof the CRISPR system are beneficial for industrial procedures that exploit bacterialcultures.
CRISPR immunity mechanism can be employed to make these cultures moreresilient to viral infection, else it can affect the productivity. Actually theinventive discovery of CRISPR immunity was discovered by scientist at Danisco,a company in the food production industry. Danisco scientists were studying abacteria known as Streptococcus thermophiles, which is used in production of cheese and yogurt. Certainviruses can affect these bacteria and also affect the quality or quantity ofthe food produced. It was revealed that CRISPR is responsible for theprotection against the viral infection. Growing further than S.thermophilus to other useful bacteria, industrialists can applythe same principles to increase lifespan of bacteria, culture sustainabilityand productivity 2,3.
2. In the Lab: Further than applications surroundingbacterial immune defences, researchers have learned how to exploit CRISPRtechnology in the lab for manipulation and create specific changes in the DNAof organisms such as, plants, mice, fish, fruit flies, and human cells. Geneare responsible for the growth and maintaining the life of organisms. A changein gene will affect the mechanisms in the organisms. CRISPR technology helps usto manipulate and modify the genes in organism in our desired interest. Scientistsfirst design and manufactures a short RNA chain which is a match to specificDNA sequence in human cells. After that as per three steps involve in CRISPRmechanism, manufactured short RNA (‘guide RNA’) attaches to CRISPR machineryand guide it to silence a gene or even change a gene sequence.
This type ofgene edition can be achieved editing sentence with a word processor to deletewords or correct spelling mistakes. This application gives us facility of makingmodel organisms with specific features and desired genetic changes also able tostudy the progress, changes and treat human disease.3.
Application in medicine: With initial accomplishments in thelab, many are seeing headed for medical applications of CRISPR technology. Oneof the unique and important application of CRISPR technology is for thetreatment of genetic diseases. The former proof that CRISPR technology can beused to correct a mutated gene and reverse the disease symptoms in a livinganimal was published in recent years. By substituting the mutated gene with itscorrected gene sequence in adult mice, scientists demonstrated that the curefor an exceptional liver disorder that could be accomplished with a singletreatment. Along with curing genetic diseases, CRISPR is also used in the domainof infectious diseases, probably providing a significant way to produce morespecific antibiotics which will kill only specific disease causing bacterialstrains whereas leaving beneficial bacteria. In recent year scientists alsostates that this technique was also used to produce white blood cells resilientto HIV infection 6,7,8Conclusions: Obviously, any freshtechnology takes roughly some period to recognise, understand and practice. Thereare a rising number of scientists from several disciplines cooperating to take aspiringCRISPR technology understanding.
As CRISPR carry on to undertake practical developments,resolving its problem associated during practice. The scenarios for thistechnologies applications continues to appear favourable and moving quickly toreality. It will be very important to understand and validate that a specificguide RNA is used to manipulate the target gene. Also it is very important to developa delivery system for CRISPR treatments for the humans before they become commonlyused in medicine field. Thoughthere are quiet several difficulties to be resolved, like delivery system, safetyconcerns, off-target effects, efficacy and ethical issues. CRISPR/ Cas9 is rapidlyused as an important means in biotechnology also in clinical practice sooner orlater. While a lot of things are about to bediscovered, but there is no uncertainty that CRISPR technology has capabilityto be a valued means in research.
Actually, there is abundant enthusiasm in thefield about this technology, there are people ready for authorize launch of someBiotech start-ups that anticipate to use CRISPR technology to cure humandiseases 8. References: 1. Palca, J. A CRISPRway to fix faulty genes.
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