Bikesh Tiwari

Roll no:09

GENETIC SCISSORS: TOOL FOR REWRITING THE CODE OF LIFE

Emmanuelle Charpenter and Jennifer Doudna won Nobel prize in chemistry 2020 for
discovering CRISPR/Cas9 genetic scissors. This scissors can be used to modify or change the
DNA of the animals, plants and microorganisms with extremely high precision. Discovery of this
technology has enhance the opportunities for the plant breeding and helps in carrying out
innovative cancer therapies. Thus, this technology might help in carrying out curing inherited
diseases in near future. CRISPR stands for Clustered Regularly interspaced Short Palindromic
Repeats. CRISPR/Cas9 is an unexpected discovery. When Emmanuelle and Jennifer were
working on immune system of streptococcus bacterium to develop new forms of antibiotics,
instead they discovered a molecular scissors that can be used to make cut in genetic material with
extremely high precision making it possible to easily change the code of life. They first showed
that CRSPR/Cas9 could edit DNA in an in vitro system in a paper published in 28th June 2012
issue of science. There discovery was rapidly expanded and soon made CRISPR a common tool
in lab around the world. Six months later, after the discovery of technology, Feng Zhang of
Broad institute showed that CRISPR worked on mammalian cells. CRISPR was also used in one
of the most controversial biomedical experiments of the past decade, when a Chinese scientist
edited the genome of human embryos, resulting in the birth of three babies with altered genes.
He was widely condemned and eventually sentenced to jail in china. Emmanuelle Charpenter
and Jennifer Doudna were originally from France and at the time of discovery worked at Umea
university. They showed they could program a small strip(guide RNA) to carry a bacterial
CRISPR-associated(cas) enzyme to exact DNA sequences, allowing them to target specific
genes. Cas then cut the double stranded DNA. In many cases DNA repairs mechanism of the
cells makes errors, which can cripple a gene- knocking out of gene in this fashion is an effective
way to study its normal role. CRISPR also allows researchers to insert a new stretch of DNA at
the cut site. There were many genome editors before CRISPR but using them was time
consuming, difficult and expensive. CRISPR works at ease with high precision and speed. The
CRISPR-Cas9 system consists of two molecules that make an edit in the DNA.

1) Cas9 – An enzyme which functions as a pair of ‘molecular scissors’. It has the ability to cut
the two strands (sense and anti-sense) of DNA at a specific location in the genome.

2) guide RNA (gRNA) – A short (20 bases or so) piece of pre-designed RNA sequence located
within a longer RNA scaffold. The gRNA guides the Cas9 to the planned part of the genome
ensuring the right place is cut. The gRNA has bases which are complementary to the target
sequence in the DNA like a very specific lego piece. This in theory ensures that the gRNA binds
only to the desired sequence and not somewhere off-target.
The Cas9 scissor enzyme is guided to the desired location and snips across both strands of the
DNA. Once this happens the cellular mechanism understand that the DNA is damaged so
initiates a repair mechanism. At this juncture it is possible to use this opportunity to introduce
changes to the genes of their design.

CRISPR-Cas9 has become popular in recent years. Church notes that the technology is easy to
use and is about four times more efficient than the previous best genome-editing tool (TALENS).
In 2013, the fiest reports of using CRISPR/Cas9 to edit human cells in the lab were published by
researchers from the laboratories of Church and Feng Zhang of the broad institute of the
Massachusetts Institute of technology and Havard. Studies using in vitro and animal models of
human disease have demonstrated that the technology can be effective in correcting genetic
defects. Examples of such diseases include cystic fibrosis, cataract and Fanconi anemia,
according to a 2016 review article published in the journal Nature biotechnology. These studies
pave the way for the therapeutic applications in human.

APPLICATION OF CRISPR/CAS9 TECHNOLOHY:

1.Generation of genetically modified mouse model of human diseases:

Traditional genetically modified mouse models are generated by transgenesis or gene targeting in
embryonic stem cells which is time consuming and expensive. The CTISPR/Cas9 system can be
used to disrupt infection of Human immunodeficiency Virus 1 in human cells, where the
expression and replication of the virus are interrupted by altering coding or non-coding
sequences during the pre-integration or provirus stage suggesting that the CRISPR/Cas9 system
may be novel therapeutic strategy against HIV. Similarly injection of plasmid containing Cas9
and SgRNA targeting the conserved regions of hepatitis B virus to tail vein, can inhibit
replication of HBV and result in the down regulation of covalently closed DNA and HBV
protein which suggest that CRISPR/Cas9 system may offer new strategy in treatment of HBV.

2. Genomic editing in specific tissue:

Using genetic scissor(CRISPR/Cas9) technology, researchers can edit the genome of

specific tissue such as the liver and brain tissues, directly and efficiently using hydrodynamic
injection and Adeno associated Virus(AAV) vectors. CRISPR/Cas9 system was successfully and
efficiently used in mammalian nervous system.

3. Flexible manipulation of epigenome:

The CRISPR system not only provides the capacity to directly modified target DNA sequences,
but also has the ability to alter the epigenome for regulating the expression of specific genes. For
example: the CRISPR system has been used to suppress DNA methyltransferases in human cells
by breaking their catalytic domains which leads to a rapid and global loss of DNA methylation
and causes cell death.

4. Novel potentiality in RNA editing:

In addition to double stranded DNA, RNA sequences can be edited using CRISPR/Cas9 system.
Such RNA editing CRISPR/Cas9 system consists of PAM presenting DNA oligonucleotide
(PAMmer), ssRNA, gRNA and a Cas9 protien.

5. In the context of psychiatry and neuropsychiatric disease, CRISPR systems have been used to
study the roles of different proteins in directing neurodevelopment, so as to better understand the
function of pathways that regulate genes and their expression.

6. Another use of this technology is the study of recurrent mutations that occurs in cancer
patients. These mutations enhance the activity of the oncogenic proteins. There impact on tumor
development can be evaluate using CRISPR/Cas9 system.

7. Food and Agriculture:

CRISPR can help food industry in 2 ways:

a. Equipping plants with resistance genes, crops can be resistant to drought, viruses, fungi
and insects. It will also reduce the reliance on pesticides and herbicides.
b. Creation of seedless fruits for sustainable food production. They can be grown from
laboratories avoiding any environmental setbacks(weather, sunlight, rain etc)
8. Production of Bio-fuel:

J. Craig Ventor(who mapped the human genome) and Exxon mobil are using CRISPR to
improve the yield of bio-fuel production from algae. After more than 8 years of research, they
successfully doubled the amount of oil produced by the aquatic organism via CRISPR gene
editing.

9. Application and implication in plant breeding :

a. Accelerate plant by allowing the introduction of precise and predictable modifications directly
in elite background

b. Multiple trait can be modified simultaneously.

c. NHEJ-mediated gene knockouts are the simplest form of targeted modification and these could
be used to eliminate genes that negatively affect food quality, to confer susceptibility to
pathogens,

d. Targeted insertion of transgenes in the fields of metabolic engineering and molecular farming.

REFERENCE:
Rodolphe Barrangou and Jennifer A. Doudna, “Applications of CRISPR Technologies in
Research and Beyond,” Nature Biotechnology, vol. 34, no. 9, September 2016, p. 937.

Liao, H.K., Gu, Y., Diaz, A., Marlett, J., Takahashi, Y., Li, M., Suzuki, K., Xu, R., Hishida, T.,
Chang, C.J., Esteban, C.R., Young, J., Izpisua Belmonte, J.C., 2015a. Use of the CRISPR/Cas9
system as an intracellular defense against HIV-1 infection in human cells. Nat. Commun. 6,
6413

DoudnaJA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9.

Science. 2014; 346:1077-87.

“The Nobel Prize in Chemistry 2020.” NobelPrize.org,

www.nobelprize.org/prizes/chemistry/2020/popular-information/.

Legal and Ethical Implications of CRISPR Applications in Psychiatry. (n.d.).

Vidyasagar, A. (2018, April 21). What Is CRISPR? Retrieved October 21, 2020, from
https://www.livescience.com/58790-crispr-explained.html