A

SEMINAR REPORT
ON
CRISPR : GENOME EDITING TECHNOLOGY
Submitted in partial fulfilment of award of
B.Tech. (CS) V Semester
By
PAKHI SHARMA
2016214

Faculty of Mathematics and Computing

Banasthali Vidyapith

Rajasthan

(July-December 2022)
 ABSTRACT

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a

component of bacterial immune systems that can cut DNA, and has been repurposed as a gene
editing tool. It acts as a precise pair of molecular scissors that can cut a target DNA sequence.
Several approaches to genome editing have been developed. A well-known one is called
CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic
repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot
of excitement in the scientific community because it is faster, cheaper, more accurate, and
more efficient than other genome editing methods.
 TABLE OF CONTENTS
1. Introduction
1.1 What is Genome editing
1.2 Why use Genome editing
2. What is CRISPR
3. Discovery of CRISPR
4. How CRISPR works
5. What can we learn from CRISPR about diseases
6. Applications
7. Merits and Demerits
7.1 Merits
7.2 Demerits
8. Future of CRISPR
9. Conclusion
10. References
 INTRODUCTION
What is Genome Editing?
Genome editing (also called gene editing) is a group of technologies that give scientists the ability to
change an organism's DNA. These technologies allow genetic material to be added, removed, or altered
at particular locations in the genome. Editing DNA can lead to changes in physical traits, like eye color,
and disease risk.

Scientists are developing gene therapies - treatments involving genome editing - to prevent and treat
diseases in humans. Genome editing tools have the potential to help treat diseases with a genomic basis,
like cystic fibrosis and diabetes.

There are two different categories of gene therapies:

 Germline therapy

Germline therapies change DNA in reproductive cells (like sperm and eggs). Changes to the DNA of
reproductive cells are passed down from generation to generation.

 Somatic therapy.

Somatic therapies target non-reproductive cells, and changes made in these cells affect only the person
who receives the gene therapy.
Why do we use Genome Editing?

Human genome editing has the potential to advance our ability to treat and cure disease, Potential
benefits of human genome editing include faster and more accurate diagnosis, more targeted treatments
and prevention of genetic disorders. Somatic gene therapies, which involve modifying a patient’s DNA
to treat or cure a disease, have been successfully used to address HIV, sickle-cell disease and
transthyretin amyloidosis. The technique could also vastly improve treatment for a variety of cancers.

Several approaches to genome editing have been developed. A well-known one is called CRISPR-Cas9.
The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is
faster, cheaper, more accurate, and more efficient than other genome editing methods.

Genome editing is of great interest in the prevention and treatment of human diseases. Currently,
genome editing is used in cells and animal models in research labs to understand diseases. Scientists are
still working to determine whether this approach is safe and effective for use in people. It is being
explored in research and clinical trials for a wide variety of diseases, including single-gene disorders
such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and
prevention of more complex diseases, such as cancer, heart disease, mental illness, and human
immunodeficiency virus (HIV) infection.
 WHAT IS CRISPR?

CRISPR is a natural part of DNA that acts as an antiviral defense mechanism, identifying and
removing specific bits of genetic info. In 2012, scientists learned that they could control what it
removes and replaces by altering a specific protein called Cas9 (CRISPR associated protein 9). This

gene editing technique is most often called CRISPR or CRISPR-Cas9.

To alter specific sequences of DNA within a cell, the CRISPR-Cas9 system needs an enzyme to edit
DNA sequences, and something to guide the enzyme to the correct place.

The enzyme which edits DNA is called Cas9, and it is often described as a pair of genetic scissors. This
is because it can cut DNA strands, allowing things to be added or removed from the ends of the cut
strands before they are re-attached.

 In order to make sure that the Cas9 enzyme cuts in the right position, it is guided by the fittingly
named guide RNA. This RNA molecule is designed to bind to a specific sequence of DNA bases and
makes sure that Cas9 only causes breaks where they are wanted.

CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an
immune defense. When infected with viruses, bacteria capture small pieces of the viruses' DNA and
insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The
CRISPR arrays allow the bacteria to "remember" the viruses (or closely related ones). If the viruses
attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to
specific regions of the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA
apart, which disables the virus.

Compared to older methods of gene editing, CRISPR offers far more precision and accuracy.
WHO DISCOVERED CRISPR: The Pioneers Behind This Technology

CRISPRs were first identified in E. coli in 1987 by a Japanese scientist, Yoshizumi Ishino, and his team,
who accidentally cloned an unusual series of repeated sequences interspersed with spacer sequences
while analyzing a gene.However, due to the lack of sufficient DNA sequence data, the function of these
arrays remained a mystery.

In 1993, researchers led by J.D. van Embden in the Netherlands discovered that different strains
of Mycobacterium tuberculosis had different spacer sequences between the DNA repeats. They
characterized M. tuberculosis strains based on their spacer sequences, a technique known as
spoligotyping. Subsequently, these sequences were identified in several other bacterial and archaeal
genomes.

Researchers Francisco Mojica and Ruud Jansen were the first to refer to them as CRISPRs in 2002. In
2005 Francisco Mojica discovered that these sequences make CRISPR an adaptive immune system.

In 2011-12 , Jennifer Doudna and Emmanuelle were among the first scientists to propose that this
microbial immunity mechanism could be harnessed for programmable genome editing and this tool has
the potential to effectively change the genetic makeup of any organism and fix a lot of problems. This
discovery changed the world of genetic engineering as previous techniques were time-consuming,
tedious, and nowhere as convenient as CRISPR.

If there’s someone who fits the term “CRISPR celebrity”, Feng Zhang is it. In 2013, his
research redirected the CRISPR focus from bacteria to eukaryotic cells. Thereafter, Dr. Zhang has done
ground-breaking work in not just advancing the technology itself, but also in expanding its applications.
 HOW CRISPR WORKS ?

 When Cas9 makes a cut in a DNA molecule, it leaves exposed ends where bases (the building blocks
of DNA) can be removed or added. This means a scientist can change the DNA sequence as they
choose.
 The Cas9 enzyme can be adapted for different functions, which allow scientists to do even more
things to the specific DNA site. One very useful adaptation involves removing the cutting function
from Cas9 and replacing it with a different type of enzyme.
 Most commonly, the new enzymes used to replace Cas9 are designed to activate (increase), or
inhibit (reduce) transcription of a specific gene. These methods are known as CRISPRa, meaning
CRISPR-activation, or CRISPRi, meaning CRISPR-interference.
 WHAT CAN WE LEARN FROM CRISPR ABOUT DISEASES

 Many diseases have a genetic element – but we don’t always know much about how the genes are
involved or which genes are behaving abnormally. When genes act abnormally, they might make a
faulty protein, or they might produce too much or too little of a protein. This can lead to a metabolic
disease such as cystic fibrosis or sickle cell anaemia. If the protein is involved in the control of the
cell cycle or apoptosis, abnormal genes may lead to cancers.
 Often CRISPR-Cas9 is used in the lab to investigate unknown functions of genes. Disrupting the
sequence or transcription of a gene allows scientists to ask many questions about how that gene
works, including:
 What happens when a gene is deleted (knocked-out)? How does a cell or model organism behave
differently when a particular gene is missing?
 What is the effect of small specific changes to a sequence? These changes, known as mutations,
often occur naturally. By recreating them in the lab we see if they have any impact on processes
within a cell, such as disrupting protein function.
 If transcription of a gene is turned up or down, does this change the characteristics of the cell or
organism?
 When studying genetic diseases, DNA-sequencing allows scientists to look at different mutations
occurring across individuals and populations. This information allows us to identify mutations found
more frequently in people with specific diseases, suggesting that the mutation increases the chance
of the disease occurring.
 To test the link between mutations and specific diseases further, scientists investigate whether
producing a specific mutation causes a cell to switch from healthy to diseased. This technique is
becoming increasingly useful in cancer research.
 The same approach is used to see if a specific genetic change causes other interesting biological
effects – for example, does changing a sequence in a bacterium allow it to become resistant to drug
treatment? This might help us tackle antibiotic resistance in future.
 APPLICATIONS
In just a few short years, CRISPR has had a massive impact on scientific research, contributing to
breakthroughs in medicine and biotechnology. Let’s take a closer look at some of the key applications of
this technology.

1. Cell and gene therapies

CRISPR is poised to revolutionize medicine, with the potential to cure a range of genetic diseases,
including neurodegenerative disease, blood disorders, cancer, and ocular disorders. As we mentioned
earlier, the first trial of a CRISPR cell therapy was performed in 2019, treating patients with sickle cell
disease. The treatment restored fetal hemoglobin, eliminating the need for a functional copy of adult
hemoglobin.

CRISPR can also be used to generate chimeric antigen receptor (CAR) T cells, a form of
immunotherapy used to treat cancer. The T cells are extracted from patients and engineered to antigen
receptors before being re-injected into the body. The receptors allow the T cells to more efficiently
target and destroy the specific type of cancer the patient suffers from.

2. Diagnostics

During the COVID 19 pandemic, CRISPR was used as both a potential therapeutics tool and as a
diagnostic tool for the coronavirus. Many diagnostics utilizing the search function of Cas9 have also
been engineered to identify other diseases, both infectious and genetic.

3. Agriculture
Gene editing technology has huge potential in agriculture, and experts suggest that CRISPR-modified
foods will be available within 5-10 years. This is primarily because it can be used to create crops that are
disease-resistant and drought-resistant. It can also be used to prolong the shelf-life of other perishable
foods, reducing food waste and allowing access to healthy foods at relatively low cost.
4. Bioenergy
As one of the leading alternatives to fossil fuels, bioenergy has been under the spotlight for a while now.
However, there are several hurdles to producing biofuels at scale. By using CRISPR, scientists have
recently been able to make some significant advances in this area.
 MERITS AND DEMERITS
CRISPR has become one of today’s most effective tools for gene-editing. CRISPR is inexpensive,
relatively simple to use and reliable, unlike other genetic engineering tools. Its success has undeniably
soared among scientists in the biotechnology industry.
While CRISPR’s advantages vary from treating genetic conditions to organ transplants, ethicists fear its
use to encouraging desired attributes rather than life-saving traits such as intelligence that could have
long-term consequences.

Advantages of CRISPR

1. Curing Genetics Disease

Provided that certain genes that causes genetic diseases have now been mapped by the science
community, CRISPR may be used to cure defective genes that cause genetic diseases.

2. Inexpensive

CRISPR-Cas9 editing is a comparatively inexpensive means of removing a gene or region or otherwise

altering it.

3. Cancer Treatments

To cure cancer, new immunotherapies may be created using CRISPR. Scientists could genetically
manipulate T-cells u sing CRISPR to find and destroy cancer cells.
4. Pest Resilient Crops

Genome editing could solve the pest and nutrition challenges faced by agriculture, particularly in the
light of climate change and population growth

5. Simple to Amend Target

Though it is not easy to set up the CRISPR-Cas9 genome editing method for the first time. It needs a lot
of grafting to refine the procedure.However once the protocol is up and running, in order to target
alternate genomic regions for editing, it is very easy to ‘chop and change the configuration.

6. Drug Research

Scientists predict that, provided the technology is relatively affordable, reliable and convenient to use,
CRISPR may theoretically speed up the drug discovery process. Some of the world’s drug
manufacturers are now integrating CRISPR technologies in their process of drug testing and discovery.

Disadvantages of CRISPR
1. Not Efficient

In any genome editing experiment, editing performance can be affected by multiple factors and can
seriously hamper your efforts. Editing performance basically represents the number of cells in your
culture vessel that have been successfully edited.

An editing efficiency of less than 100% is by no means a failure, but it does mean that the findings need
to be closely viewed. The unedited cells inside your population can mask any subtle impact of your
editing
2. Changes to the Germ-Line

A basic distinction needs to be made in medical applications between improvements to body cells and
modifications to germ-line cells such as egg and sperm cells. The next generations are inherited through
changes to the germ-line. However, it is not possible to predict the long-term consequences of this type
of germ-line treatment. Ethicists are anxious that other characteristics, such as higher intelligence or
larger muscles, may now be transferred into the genome if genetic abnormalities can be reversed at the
embryonic level. How far from “designer humans” are we then?

3. Time Consuming

Not all laboratories have an existing pipeline for genome editing. If you are in a laboratory without such
a pipeline but have established CRISPR-Cas9 genome editing as the perfect technique to further your
research, then chances are that you will have to build and refine the protocol. Optimizing a protocol for
CRISPR-Cas9 can be difficult and time consuming.

4. Off Target Effects

The CRISPR-Cas9 method is extremely precise in principle, but in reality not so much. It can cause
mutations elsewhere in the gene, known as off-target’ modifications. Off-target impacts are random and
can affect other genes or regions unduly.
 FUTURE OF CRISPR

There can be no doubt that CRISPR-Cas9 has revolutionized the field of genome engineering. However,
we’re only just beginning to see the benefits and possibilities of this incredible technology - with a
variety of successful preclinical studies and more clinical trials being approved, the dream of curing
human disease by editing our DNA is now very real.

There is huge potential for CRISPR to be used as a treatment for disease, not just a tool for studying it.
In future CRISPR could be used in embryos to change mutations known to increase the risk of disease.

By editing the DNA of crop plants, CRISPR will help create crops which produce a lot more food, or
become resistant to drought. These increased yields will help overcome shortages of food, agricultural
land, and water.

While we are still in the early years of clinical trials, this technology could be used to treat thousands of
genetic conditions in the future, including breast and ovarian cancer linked to BRCA mutations, beta-
thalassemia, cystic fibrosis, and early-onset Alzheimer’s and many more.

There are also an increasing number of biotech startup companies focusing on CRISPR-Cas9 gene
editing technology, and many researchers are continually finding new ways of applying this technology
to solve real-world problems, including epigenome editing, new cell and gene therapies, infectious
disease research, and for the conservation of endangered species.
 CONCLUSION

Thanks to its pinpoint accuracy and relatively low production costs, CRISPR could potentially change
everything involving genes: from curing diseases and improving agriculture, to repairing genetic
disorders like sickle cell anemia or hemophilia. Many scientists believe it could lead to breakthroughs in
the fight against cancer, HIV, and heart disease. Some hope it may even lead to a cure for aging itself.
Scientists around the world are conducting numerous CRISPR trials, trying to turn this incredible
potential into reality. While those studies are ongoing, some of them have already shown results. For
example, in 2017, CRISPR was used to treat a genetic heart defect in human embryos.
Few celebrities, such as Bill Gates and John Oliver, have championed the positive potential of CRISPR.
On the other hand, movies like Rampage (2018) starring Dwayne “The Rock” Johnson use scientific
inaccuracies to allow for a plot that gets out of hand in a way that CRISPR scientists have not, and
would not do .
No one knows when CRISPR’s potential will be fulfilled. There are currently numerous organizations
and scientific institutions studying CRISPR, and it will take a lot of trial and error to make complex
genetic changes. But with every passing day, science advances a little bit farther.

One day, CRISPR may cure diseases and disorders that have plagued humankind for centuries. It could
also bring up ethical questions that we’ve never had to face before. Whenever that day comes, it could
very well change the world.
 REFERENCE

1. https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/
2. https://www.synthego.com/learn/crispr
3. https://www.genome.gov/about-genomics/policy-issues/what-is-Genome-Editing
4. https://edu.gcfglobal.org/en/thenow/how-crispr-could-change-the-world/1/
5. https://www.yourgenome.org/facts/how-do-we-use-crispr-gene-editing-to-study-diseases/
6.