CRISPR lot of attraction in the scientific community. Researcher

CRISPR
technology in research and beyond:

Abstract: 

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In last few years a revolutionary technology
has gained a lot of attraction in the scientific community. Researcher around
the globe have adopted this new technology that facilitates making specific manipulation
of desired DNA of humans, animals, microbes and plant. As compared to prior
techniques used for the modification of DNA, this new technology is very much easier
and rapid. This technology is called as “CRISPR” (Clustered Regularly
Interspaced Short Palindromic Repeat) and this technology not only help us
easier way to conduct research procedures but also it helps in curing the
diseases. Clustered Regularly Interspaced Short Palindromic Repeat this name
itself states the distinctive organization of short and moderately palindromic
repeated 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 the
above organisms. As we know immune system responsible for the protection of the
living organism’s health. Like all other living animal, bacterial cells can be attacked
by viruses. Which are small, contagious agents. If a virus causes an infection
to the bacteria, the CRISPR immune system of that bacteria gets activated and
protect it from that virus by killing it. CRISPR immune system destroys the
genomic material of that virus so that it cannot replicate itself and harm the host
cell. Only just, CRISPR related protein 9 (Cas9) has been used as an important means
in the field of biotechnology 1,2,3,4.  

Introduction:

CRISPR gene editing is taking biomedical research by storm. Providing
the ultimate toolbox for genetic manipulation, many new applications for this
technology are now being investigated and established. CRISPR systems are
already delivering superior genetic models for fundamental disease research,
drug screening and therapy development, rapid diagnostics, in vivo editing and
correction of heritable conditions and now the first human CRISPR clinical trials. The
continuing patent battle for CRISPR-Cas9 licensing rights and the emergence of
new editing systems such as Cpf1 has so far done nothing to slow the advance of
CRISPR-Cas9 as the leading gene editing system. There are weekly press releases
and updates on new advances and discoveries made possible with this technology;
the first evidence is now emerging that CRISPR-Cas9 could provide cures for
major diseases including cancers and devastating human viruses such as HIV-1

 

The key to CRISPR-Cas9’s uptake is its ease of application
and design, with retargeting only a matter of designing new guide RNA. It has
quickly surpassed TALENs (Transcription Activator-Like Effector Nucleases) and
ZFNs (Zinc Finger Nucleases) where editing, now possible with CRISPR, was
previously prohibitively complex and time-consuming. As well as correcting gene
mutations with scar-less modifications, with CRISPR-Cas9 it is possible to
control the expression of entire genes offering longer term expression
alteration compared to other methods such as RNAi. CRISPR-Cas9 systems,
tools and basic methodology are very accessible as ready to go toolkits that
anyone with lab space and an idea can pick up and start working with. This is
thanks largely to the efforts of Addgene and
commercial service and product providers. Alongside CRISPR research there are
innovations in companion technologies and design software. In response to a
growing need, companies such as Desktop Genetics have
developed open access software to accelerate CRISPR experimentation and analysis.

 

Mechanism:

Figure 1: The steps involved in CRISPR
mechanism.

CRISPRs are sections in the
genome of bacteria which helps to protect against viral infection. These
sections constitute short DNA repeats (black diamonds) and spacers (coloured
boxes). When a formerly unknown virus infects bacteria, a new spacer from the
virus is merged between current spacers. The CRISPR system gets activated, it
transcribed and proceed to produce short CRISPR RNA molecules. The CRISPR RNA
links with and guides the bacterial CRISPR machinery to an identical target
sequence in the infecting virus. The CRISPR machinery cut and destroy the
infecting virus genome. These
spacers are derivative of virus DNA that have previous infection to the host
bacteria. Therefore, spacers work as a ‘genetic memory’ of earlier infections.
If another infection by the previous same virus occurs, then the CRISPR system
will destroy any similar spacer sequence and will defend the bacteria from
viral infection. If a totally new virus infects the bacteria, then new spacers
will produce and incorporated in the chain of spacers. The CRISPR system
protects 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 that
are inserted into CRISPR sequence as new spacers.

Step 2. CRISPR RNA Production:
CRISPR sequence and spacers in the bacterial DNA sequence go through
transcription. This RNA chain is spliced into short fragments called CRISPR
RNAs.

Step 3. Targeting: CRISPR
RNAs guide bacterial CRISPR machinery to kill the viral genetic material.
Because CRISPR RNA sequence are copied from the sequence of virus DNA during
the process of adaption. They are precise matches of the viral genetic material
and therefore acts as excellent guides.

The specificity
of CRISPR immunity mechanism in identifying and killing infecting viruses is
not just beneficial for bacteria. Inventive applications of this primeval yet graceful
protection mechanism have arisen in disciplines as various as medicine,
research, and industry.

Applications
of CRISPR technology:

1.    
In Industry:
The integral functions
of the CRISPR system are beneficial for industrial procedures that exploit bacterial
cultures. CRISPR immunity mechanism can be employed to make these cultures more
resilient to viral infection, else it can affect the productivity. Actually the
inventive discovery of CRISPR immunity was discovered by scientist at Danisco,
a company in the food production industry. Danisco scientists were studying a
bacteria known as Streptococcus thermophiles, which is used in production of cheese and yogurt. Certain
viruses can affect these bacteria and also affect the quality or quantity of
the food produced. It was revealed that CRISPR is responsible for the
protection against the viral infection. Growing further than S.
thermophilus to other useful bacteria, industrialists can apply
the same principles to increase lifespan of bacteria, culture sustainability
and productivity 2,3.

2.    
In the Lab: Further than applications surrounding
bacterial immune defences, researchers have learned how to exploit CRISPR
technology in the lab for manipulation and create specific changes in the DNA
of organisms such as, plants, mice, fish, fruit flies, and human cells. Gene
are responsible for the growth and maintaining the life of organisms. A change
in gene will affect the mechanisms in the organisms. CRISPR technology helps us
to manipulate and modify the genes in organism in our desired interest. Scientists
first design and manufactures a short RNA chain which is a match to specific
DNA sequence in human cells. After that as per three steps involve in CRISPR
mechanism, manufactured short RNA (‘guide RNA’) attaches to CRISPR machinery
and guide it to silence a gene or even change a gene sequence. This type of
gene edition can be achieved editing sentence with a word processor to delete
words or correct spelling mistakes. This application gives us facility of making
model organisms with specific features and desired genetic changes also able to
study the progress, changes and treat human disease.

3.    
Application in medicine: With initial accomplishments in the
lab, many are seeing headed for medical applications of CRISPR technology. One
of the unique and important application of CRISPR technology is for the
treatment of genetic diseases. The former proof that CRISPR technology can be
used to correct a mutated gene and reverse the disease symptoms in a living
animal was published in recent years. By substituting the mutated gene with its
corrected gene sequence in adult mice, scientists demonstrated that the cure
for an exceptional liver disorder that could be accomplished with a single
treatment. Along with curing genetic diseases, CRISPR is also used in the domain
of infectious diseases, probably providing a significant way to produce more
specific antibiotics which will kill only specific disease causing bacterial
strains whereas leaving beneficial bacteria. In recent year scientists also
states that this technique was also used to produce white blood cells resilient
to HIV infection 6,7,8

Conclusions:

Obviously, any fresh
technology takes roughly some period to recognise, understand and practice. There
are a rising number of scientists from several disciplines cooperating to take aspiring
CRISPR technology understanding. As CRISPR carry on to undertake practical developments,
resolving its problem associated during practice. The scenarios for this
technologies applications continues to appear favourable and moving quickly to
reality. It will be very important to understand and validate that a specific
guide RNA is used to manipulate the target gene. Also it is very important to develop
a delivery system for CRISPR treatments for the humans before they become commonly
used in medicine field. Though
there are quiet several difficulties to be resolved, like delivery system, safety
concerns, off-target effects, efficacy and ethical issues. CRISPR/ Cas9 is rapidly
used as an important means in biotechnology also in clinical practice sooner or
later. While a lot of things are about to be
discovered, but there is no uncertainty that CRISPR technology has capability
to be a valued means in research. Actually, there is abundant enthusiasm in the
field about this technology, there are people ready for authorize launch of some
Biotech start-ups that anticipate to use CRISPR technology to cure human
diseases 8.

 

References:

1. Palca, J. A CRISPR
way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes>
29 June 2014

2. Pennisi,
E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. Barrangou, R., Fremaux,
C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and
Horvath, P. (2007). CRISPR provides acquired resistance against viruses in
prokaryotes. Science 315, 1709–1712.

4. Brouns, S.J., Jore, M.M.,
Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J.,
Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Small CRISPR RNAs
guide antiviral defense in prokaryotes. Science 321, 960–964.

5. Barrangou, R. and Marraffini,
L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014).
Molecular Cell 54, 234-244.

6. CRISPR
reverses disease symptoms in living animals for first time. (31 March 2014).
Genetic Engineering and Biotechnology News.
27 July 2014

7. Pollack, A. A powerful new way to edit
DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0>
16 July 2014

8. Gene editing technique
allows for HIV resistance?   13 June 2014

9. Yin, C., Zhang,
T., Qu, X., Zhang, Y., Putatunda, R., Xiao, X., … & Qin, X. (2017). In
vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in
animal models. Molecular Therapy.) 

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Ajetunmobi A, Dillard V. Guide Picker is a comprehensive design tool for
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2017 Mar 14;18(1):167.

11. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M.,
Makarova, K. S., Essletzbichler, P., … & Koonin, E. V. (2015). Cpf1 is a
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759-771.

12.
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Lopez, J. M., … & Joung, J. K. (2016). Genome-wide specificities of
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