SUBMITTED BY ROLL NO.

91-105

SUBMITTED TO: DR. WAQAR SALEEM

TOPIC: CRISPR-CAS 9 system

Introduction:
Crispr is abbreviated as Clustered Regularly Interspaced Short Palindromic
Sequence. This technology is used for editing genome into our required shape. It
helps the researchers in editing the DNA and giving it the desired form. It has a
wide range of application like making the wrong or defective gene into right one,
treating and in the prevention of disease.
Discovery:
It is believed that CRISPR has been discovered independently in three parts of
world. Firstly, it was discovered by a Japanese researcher Yoshihito Ishan and his
colleagues at the Osaka University in 1987. They cloned their target gene with
the CRISPR system and tis was completely coincidental. At that time, they did not
name it as CRISPR. Secondly, it was discovered in 1993 by the scientists who were
working on MYOBACTERIUM TUBERCOLOSIS in Netherlands. They observed
interrupted direct repeats in that bacterium. They knew the variety of sequences
that intervened the direct repeats. They discover a method of gene typing by this
that was named as polytyping, which is still in use. At the same time, repeats
were seen in some old species of bacteria like Halofuran and Heliacal species and
it had been studied in University of Alicante in Spain.

How CRISPR-CAS9 system works?

CRISPR-CAS9 was adapted from a naturally occurring genome editing system in
bacteria. CRISPR system is an RNA mediated nuclease system. The bacterial RNA
captures invading DNA and show enzymatic activity and breaks down the DNA
into shorter fragments. Bacterial RNA recognizes viral DNA due to some specific
sites called PAM (photo spacer adjacent motif). The RNA binds with these sites
and CAS enzyme arrives and break down the DNA into shorter fragments. CRISPR
is a specialized region of with two different properties: presence of nucleotides
and spacers. In case of bacteria, spacers are taken from previous invading viruses.
They make memories about that virus so that they can fight later with that
bacteria. Like in case of streptococcus thermophilus bacteria that is commonly
found in dairy products. Scientists observed that when virus was incorporated in
bacteria, they developed spacers against them in the CRISPR region. Moreover,
the DNA sequence of the spacers were identical to viral genome.
CRISPR RNA (crane): Once a spacer is incorporated and virus attacks again,
CRISPR is transcribed and it is converted into crane or complementary sequence
or single stranded RNA. Each CRISPR RNA consists of a spacer and a nucleotide.
CAS9: The Cas9 protein is an enzyme that cuts foreign DNA.
The protein typically binds to two RNA molecules: crane and another called trans-
crane. The two RNA then guides the nuclease to the target site where it will cut
the foreign DNA. The expanse of DNA is complementary to 20 nucleotide cranes.
Using two separate regions Cas9 cuts the both strands of DNA double helix known
as Double stranded break. There is a self-defensive mechanism that ensures that
Cas9 doesn’t cut the genome. Short DNA sequences knowns as PAMs serves as
tags and sit adjacent to the target DNA sequence. If they don’t find PAMs on DNA,
 they won’t cut it. This is the only reason why Cas9 don’t attack the CRISPR.

CRISPR-CAS9 as a genome editing technique:

The genome of various organisms a series of messages and instructions within
their DNA sequence. Genome editing involves changes those sequences thereby,
changing that messages. This can be done by inserting a cut or break in the DNA
and tricking a cell’s natural DNA repair mechanism into introducing the changes
one wants. CRISPR-CAS9 is capable to do this. In 2012, two research papers were
published. The study conducted by separate groups concluded that CAS9 can cut
any desirable region of DNA. This can be done by simply changing the sequence of
crane into the desirable sequence which binds to its complementary sequence of
DNA. There is another sampling of this system that is by fusing trac-RNA to create
a single guide RNA. Thus, genome editing requires only two components that is
guide RNA and CAS9 protein.
Operationally, a stretch of 20 nucleotides base pair RNA is designed that matches
the gene, one wants to edit. An RNA molecule complementary to 20 base pairs is
prepared. It is important that RNA nucleotide sequence must resemble with the
target gene and nowhere in the genome. Thus, RNA plus the Cas protein will cut
the DNA like scissors.
Once, the DNA is cut the cell’s natural repair mechanism kicks in and work to
introduce mutations and other changes to the genome. This can happen in two
ways:
Non-homologous end joining: This method involves the gluing of the two cuts
back together and this method tends to introduce errors. Some important
nucleotides accidentally deleted or inserted resulting to defective gene.
Second method: In this method, the break is filled by fixing a nucleotide in the
gap. In this, scientists used a short strand of DNA nucleotide as a template.
Scientist can apply the DNA of their own interest and this method is very helpful
in repairing the defective genes. In this method, you cut the gene at the desire
region and put the beneficial DNA fragment. This method is used for curing the
disease that are mostly inherited.

CRISPR CAS9 beyond genome editing

Regulate gene expression: Dead CAS9 can be recruited to desirable DNA
sequences as a fusing protein with the transactivation or transrepression domain
of a transcription factor.
Visualize selected loci in the living cells: Dead CAS9 can be fused with a
fluorescent protein to visualize 3D architecture of the genome.
Uses of CRISPR CAS9
CRISPR-CAS9 system has become very popular in the recent years. It has been
seen that this system is four times more efficient than other genome editing tools
(also called Talen’s).
In 2013, this technique was used for the first time to edit a human cell genome by
the researchers at the laboratory of Church. Studying using in-vitro and animal
models of human diseases have shown that this method is effective in correcting
genetic defects. Example of such diseases include cystic fibrosis cataracts and
Fanconi anemia.

Advantages of CRISPR-CAS9 system

CRISPR technology has also been applied in the food and agriculture industries to
engineer probiotic cultures and to vaccinate industrial cultures (like yogurt)
against other microbes. It is also been used in crops to increase yield, drought
tolerance and nutritional purposes. One other beneficial point of this method that
we can create gene of our desirable shape. These are some methods which
enhance the chance of inheritance of a particular gene from to parents to
offspring. Finally, generation after generation, the specific characters spread
through the entire population. Gene editing can be very helpful in the prevention
of disease such as by sterilizing the vector malaria we can easily control malaria
from spreading. Moreover, gene editing can also be used for the destruction of
many trespassing animals and to delete the resistances of many pests and
herbicides.
Simple,accessible,cheap and more versatile than ZFNS and TALENS.
Create genetic variants for a desired locus.
CRISPR-CAS9 also allows multiple gene editing by the simultaneously expression
of two or more sgRNAs.
Allows the inexpensive assembly of gRNA libraries so that this system can be used
for high throughput functional genomic editing within the budget of any
molecular laboratory.
This system can introduce biallelic or homozygous mutation directly in the first
generation.
Being an easy and affordable tool, this system promises revolutionize basic and
applied plant research.

Human disease modelling

Cancer study: Numerous excellent studies have been introduction with the aim
of production of in vivo and in vitro cancer models, recently it has been reported
on the production of pancreatic cancer by automatic addition of many gene
networking sets using transection based multiplex delivery of CRISPR CAS9
system’s components to the pancreas of adult mice.
Neurological Disease Modelling: Certain high quality studies define the
production of nuerological models. Two very good examples of nuerological
modelling includes the use of CRISPR CAS9 system in an iPSC-based model to
explain the cause of epilepsy by SCN1A loss of function mutations.
Cardiovascular Model: CAS9 mice robustly express high levels of CAS9 mainly in
heart cells after the insertion of CAS9 via injection. As a proof for this experiment,
the researcher used AAVs to deliver sgRNAs against Myh6 and demonstrated
cardiac specific genome driving at the Mhy6 locus. A reduced in blood cholesterol
level was observed in the mice.

Applications and implications in plants

Accelerate plant breeding by allowing the introduction of precise and predictable
modifications directly in an elite background.
Multiple traits can be modified simultaneously.
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
susceptibility to pathogens.
The insertion of large sequences by NHEJ or HR would allow the introduction of
transgenes at defined loci that promote high level transcription and do not
interfere with the activity of endogenous genes.
Allow targeted molecular trait stacking.
Plants altered by the excision or insertion of a few nucleotides using CRISPR CAS9
would not be classified as a genetically modified organism.
Targeted insertion of transgenes in the field of metabolic engineering and
molecular farming.

Drawback of CRISPR CAS9 method

One of the few criticisms of the CRISPR CAS9 system is the relatively high
frequency of off-target mutations reported in some of the earlier studies.
The limited data thus far available suggest that off far target effects are rare in
plants, which can be eliminated by back crossing.
Off target effects vary often among different cell types in the same species.
According to Church the biggest limitation of this system is that it isn’t 100%
effective. In addition, the gene driving can also be changed. According to a article
conducted in 2014 by two scientist in rice, gene driving occur only in half of
thecell that were given with RNA CAS9 complex. While many other experiments
have shown that if we change the target, efficiency can be increased to 80% or
more.
There is another phenomenon which is called off target gene sequencing in which
RNA cut the DNA not at the desirable site but other than that. This can cause
many defects unintentionally. Additionally, the scientists also noted that even
when the CAS system cut the DNA on its own, it can also not provide us with a
precise edit. The scientists called this as gene vandalism.

Strategies to reduce off target genome editing

Mutated version of CAS9 protein has also been manufactured with D10A defect in
RUVC nuclease domain that converts it into a nickase.
If we use two CAS9 nickase, it can therefore produce offset single strand nicks
that produce a staggered DSB.
Potentially capable off-target sites were not likely to be enough close to each
other to enable more than individual nicks to work.

Setting limits
The so much benfits of CRISPR technology raises many question about the moral
merits and results of dealing with DNA.
Scientist pointed out the ecological effects of gene editing system. A self
introduced gene can spread behind the limit which is the result of crossbreeding.
Gene editing can also minimize the genetic variety of the targeted gene. Process
of modification of human’s embryo as well as sex cell like sperm cell is called as
Germline gene driving . Because if we change these cell, the changes can be
passed on to the next generations, using CRISPR technology to make modified sex
cells have raised a lot of ethical issues.
Changeable efficiency, target deviated effects and unpreciable edits all pose
safety issues. Moreover, there are still many effects that scientist are not familiar
with. According to a scientific study, Germline gene driving may increase the
possibility of genetic defects in the future generation which happens purely
unintentionally. This is because we have certain limits of knowledge about human
genetics and gene environment interactions and how the disease occurs including
the relation of one disease in a patient with the other disease with the same
patient.