NAZYL HEINS M.

GUEVARA
BS ECE 1-5
 The voyage of mankind for further understanding the circle of life, we were able
to dive into various phase of mankind’s nature of existence from life, maturity,
functionality, and death itself. The discovery of the cell by Robert Hooke enables
prodigies of today to make significant scientific advancements transcending humanity
perception’s into establishing a rational view towards our existence (NatGeo,2019).
Prior to this discovery, scientist further strengthen the principle theory of cell as the
fundamental unit of living organism when the era of cytogenetics begins to shift
mankind’s idea into the study of genetics catering the foundation of life. Within the cell,
it holds the pillars of life from its essential structure, nucleus as the cell control center
that upholds the capability for tailoring the constant process of life. It also houses the
thread-like chromatins made up with the thousand string genes compromised of
chemical substance known Deoxyribonucleic Acid (DNA). This genetic material acts as
the fundamental concept of Mendel’s theory of inheritance wherein it was responsible
for the small variations and uniqueness of each living organisms.

1. What is CRISPR?

Genetics and Genome Engineering had been a sensational field of

scientific discipline catering further studies on the book of life in order further
understand the cycle of life from birth, living, reproduction, and death.
According to Ran (2013), biological system engineering carries a great
potential application across biotechnology, and medicine leading into an
evolutionary birth of scientific methods regarding the technical capabilities of
altering an organisms’ DNA known as Genome Editing. This method
resurfaces the potential technique along with emerging technologies for
humanity to achieve the apex of success with Genetic Modification and
Variation. As stated by the National Health Institute, Genome Editing
imposed a prominent transition humanitarian power to set forth the venture
towards the understanding the mechanism of basic unit of life, how and why
organisms innate traits carried by the DNA could be modified as per desired
of the person itself. Moreover, this process had been recognized for its
probable contribution in medicine where it imposed the ability for treatment
as well as to combat the risk of acquiring diseases.
The preceding journey of discovery towards genetic modification lead into
the innovation of a profound genome editing tool called as the CRISPR Cas9
system, remarked by various health organization as a simpler, faster,
precise, and efficient DNA sequencing method within a living cells. The
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9
system was recognized as an adaptive immune system naturally exist on
microbes such as bacteria having a defense mechanism on viruses through
targeting its DNA (Cong, 2015). Immediately upon virus attacks into a living
organism like a bacterium, it rouses its ability to detect these dreadful
parasites enabling them to solidify their response through generating two
types of RNA, one has a similar sequence that matches the invading virus.
Moreover, this method encompassed of three interlinked classes, the highly
functional Class II system dominantly acts as the pillar of modern genetic
science which shelters the overall working mechanism of RNA necessary for
the formation of the CRISPR Cas9 endonucleus crucial for the protection of
cells into invading foreign DNA (Markova, 2011). From the class II system,
two RNA are generated the single RNA guided which was combined with
Cpf1; and the other one was the single protein effector or the tracrRNA,
carried to form a complex bond with the protein, Cas9 wherein this
phenomenon occurs on the abovementioned bacterium response. This
astonishing method was possible through this RNA Guided Cas9 nucleus
targets pairing a specific sequence from the existing DNA structure. But how
does it disable the existing DNA? Prior to this succeeding process, the
combination of the tracrRNA and Cas9 protein inside the nucleus sequenced
into the linearized plasmids, they will lock into the short and precise proto-
spacer adjacent motif (PAM) sequence in the DNA, acts as the key feature of
the editing process. Intensifying this voyage of discovery, they also found out
the definite Cas9 activity encompassed a viral DNA cut into a precise position
to the PAM in order to crRNAs strands completion (Horvath, 2008)
Over the past years of continuous study of human diseases, researchers
discovered the function of genes as the blueprint of life wherein it has the
ability to mutate, once it imposed threat of disease to the parent cell from a
haploid latter, into a diploid cell. For this reason, researchers adhere on the
potential contribution of this method for the treatment of diseases since,
mostly our genes sheltered the traits including diseases from our parents,
also known as genetically innate diseases (Ding, 2012).
In research trial, researchers disabled a gene using the mentioned
system, wherein it was similarly compared to a scissors that cuts the DNA
sequence in order to replace with the carried RNA of the CRISPR – Cas9
enzyme (Cyranoski,2016). It’s promising results was significantly founded by
its programmable ability as target- specific nucleus enables it recognized the
defected part within the sequence that needs treatment and alteration of new
protein strands. CRISPR CAS9 System loads the new DNA Sequence into
the new one leaving a strands breakage where the working mechanism of
cell repairs it for the completion of the modified DNA Sequence. Furthermore,
the disabled code sequence of the DNA along with the PD-1 that naturally
regulates the cell’s immune response was discovered to be an essential by
product of this methods. Wherein positively, without the PD-1, the body’s
defense mechanism strengthens the cells armor to strike down cancers cells.
This revolutionary transition in Genetic engineering opened the gateway
for opportunities for cure of diseases as well as upholding the capability to
modify specific traits for a specific organism. Its deployment acts primary
 facilitator of the modern genome sequencing of eukaryotic cells offering a
wide variety of applications from biotechnology, agriculture, medicine, and
science of living itself.

2. What is the difference between CRISPR and restriction

enzymes?

Gene modification are primarily one the endeavor of the field of

Genetics and molecular biology through the genetic engineering. This
method plays a crucial role in today’s world where genes resurface the idea
of its existence to the overall function of genome living cells. Genome editing
encompassed of various techniques used in modern times for mankind’s
deep comprehension towards science of living. Such as the CRISPR CAS9
system and Restriction Enzymes are well known practices in gene
modifications.
First, the Restriction Enzymes have been a major element throughout
mankind’s continual discovery on DNA operations and technology. It
significantly paved the way of scientist towards the possibilities for discovery
of the underlying composition of fundamental unit of one’s living leading into
the manifestation of various method of genome editing. The notion of these
restriction enzymes was first introduced by the Swiss Microbiologist Werner
Arber through his experimentation on a host controlled restriction of
bacteriophages- defined as viral particle which has the ability to replicate
their own DNA. Latter, various studies supported Arber’s theory wherein
indicates some of the phages were controlled by their host causing the
growth to decline since, enzymes were able to broken down its DNA (Luria,
1952). In the contrary, he was able to strengthen the idea that other
bacteriophages undergo growth and development when it previously has
been contact with the same bacteria causing bacterial strain to fully flourish
capable of infecting new host cell. Generally, these restriction enzymes were
responsible for the endonucleolytic scission as endonuclease R observed
through isolating the enzyme of Escherichia coli K (Meselson & Yuan, 1968).
This was followed by the first experimentation of Danna and Nathans (1971)
using the restriction enzyme known as the endonuclease R generating specific
fragments of simian virus SV40 DNA allowing them to significantly
accomplished the goal if restricting the endonuclease from Hemophilus
influenza.
This method had been instrumental molecular biological techniques.
Whereas these enzymes recognize specific sequences of DNA necessary for
the cleaving process of the strands occurring on particular place called the
restrictions sites. Restriction enzymes create incision to the restriction points
ends through the sugar phosphate backbones of the DNA strands. Disabling
the both interlinked plasmids in the sequence, DNA molecules was chopped
into varying fragments for enzyme digestion through the application of Ulrich
Loenings’s polyacrylamide gel electrophoresis commonly used in separating
RNA species (Roberts, 2005). Although these enzymes identify specific DNA
sequences, unfortunately it’s a difficult process for this advancement to exactly
determine the insertion point. Moreover, these enzyme was not capable of
cleaving DNA randomly since rendering these immensely depend on the
external condition so it’s unsuitable for use as cloning and mapping reagents.
Aside from this, Danna and Nathans made prominent discoveries on the
potential utilization of these fragments to establish a rough map of the
genome as well as the specific origin of restriction points where the incision
and replication take’s place. Therefore, the restriction enzymes established
an insightful information of restriction analysis due to its specific manner
enabling scientist to retrace its occurrence.
On the contrary, CRISPR was the revolutionary genetic technique in
modern society which offers the promising cure for any genetic disease
through its advance mechanism for gene editing. It imposed a natural
mechanism commonly occur on bacteria derived the adaptive immune
response against the foreign genetic elements affecting host cells. These
techniques employed an extremely precise cuts on the genome sequence.
Furthermore, the produced RNA formed a complex bond with the CAS9
effector that act as guided crRNA/tracRNA. Once the formation process was
achieved, the Cas9 nucleus disable the programmed defected within a
particular DNA sequence (MacDonald,2016). Its incredible preciseness and
efficiency enable scientist to easily manipulate genetic codes thus, upholding
its prominent contribution to combat human genetic diseases. Recent studies
coagulate this claim through performing several trials to examine its potential
roots for the treatment of the vile cancer cell.
Both genome modification system rapidly geneticizes the blueprint of our
life needed for transcribing the traits within complementary stands. These
strands essentially act as the pillars of the concepts of inheritance and
variance with living organism specifically human beings. Abovementioned
methods employed its functionality of clipping its target DNA sequence which
primitively established and shared trait within the cells comes from its parent
cell. However, CRISPR imposed a defined and accurate specification on
infected DNA rather than the restriction enzymes.
3. What are your insights regarding the discovery of CRISPR-
CAS 9?

CRISPR-Cas 9 had been a sensational tool in the field of Genetics and

Molecular biology. It’s birth along with the other commonly employed genetic
modification techniques lead mankind into a prominent breakthrough of
building their intuition towards the relativity of existence, science, and
technology. The field of Genetic Engineering was able to gradually built its
name into the society through these emerging biotechnological process and
concepts introduced into humanity. Existing scientific process on the other
hand, enables one to recognized the truth what science has to explains
supported and solidify by these existing variables. The variables are
interlinked with one another thus, it notably established a rational concept
and a veracious view towards our reality.
CRISPR CAS9 was known to be the modern’s days go to genome editing
tool since, it was easier, simpler, and faster method (Fernandez, 2021). Over
the past years, scientist shared this innovative system to the society offering
a hope this technology holds a great power to treat human diseases
specifically the genetically innate illness. Prior to its undergoing process
mentioned previously, CRISPR CAS9 system could disable the infected
genetic mutation within an existing cell with new and complex form proteins
strands of RNA and Cas9 nucleus that acts like a scissor. This alteration was
remarked by researchers as an immune response mechanism to generate
and repairing disease mutated cells that firmly starts with slicing its target
sequence for replacement of the guided RNA (Kaiser, 2021).
Presently, these CRISPR system showed its crucial role to the field of
healthcare and biotechnology wherein it was able to elevate the development
of Covid-19 vaccine. While the world was amidst of this COVID-19 pandemic,
the genetic tool widely made a significant contribution to further determine
and uncover the underlying capabilities of these guided RNAs. Since, this
biotechnological system was developed to have a naturally adaptive immune
response, it has the ability to attack new variants of viruses (Isaacson, 2021).
The new wave of viral infection was endeavor to be another triumph of the
CRISPR system. Moderna and Pfizer- BioNtech companies are the leading
producers of SARS-COV-2 vaccines which rely on the undeniable molecular
property of RNA due to its broad part in the prevention and disease control
such as for cell anemia, polio, and other diseases.
For this instance, these two vaccine companies pioneer to work through
the mRNA technology adapted into the CRISPR CAS9 system technique.
Wherein these programmable mRNA vaccines prompt antigens to stimulate
the body’s immune response resulting to manifestations of formation of
complex enzymes, capable to detect viral infections and transcribed genes
in order to recreate new and well-conditioned protein strands. It was stated
 the Abbott (2020), Cas9 tends to have its relative counterpart for the SARS-
COV-2 sequence known as Cas13d necessary for the envisioned
development of PAC-MAN (prophylactic antiviral CRISPR) found in human
cells produced to attack the inhibiting viral infections including their viral RNA
like the SARS-COV-2 and Influenza viruses.
Undeniably, CRISP CAS System was a truly influential biotechnological
process whereas it profoundly built its role into the society as a hope for life.
This method was able to shed light into the future of humanity where genetic
modification would be a common phenomenon to save lives Emerging
scientific studies and technology offers an easily and precise control over
antigen living with us. Crucially, mankind awareness harnesses them to take
part into this global and public health issue towards this war against the
unseen enemy tormented lives of many, and shutting down functional
community.

4. Describe the mechanism of CRISPR in layman's term.

Genome editing tool CRISPR Cas9 system was one of the mainstream
technique in the contemporary society due to inevitable advancement
mankind pursue. Furthermore, it’s operation was absolutely prevalent into
the current menace the healthcare system and global community had been
battling. As humanity thrives to surpass the challenge of this social dilemma
brought by Covid-19 virus, CRISPR Cas9 system was one of the key role
players that helped humanity to walk into this transience founded with the
structured backbone of the COVID-19 facts such as its treat and prevention
(Cross, 2021). Moreover, this technique was previously applied in various
experimentations and trials in order to address the need for control and
prevention on occurring human disease, commonly genetically innate
diseases. But what exactly is the CRISPR System and how does it function
within the body, particularly within our cells?
The CRISPR Cas9 system was natural prokaryotic adaptive immune
system response that targets precise DNA sequence for genome
modification. This was done through the existence of two RNA which forms
a complex interconnection with Cas9 enzymes latter, combined to generate
the Ca9 nucleus that carries the guided RNA. This technique was similarly
compared by Jennifer Doudna, one of the creators of this revolutionary
Genome Modification Tool with a scissor wherein the RNA guided nucleus
that cut its target points within the DNA sequence in order to replace the
defected DNA sequence in a cell with the RNA inside the Cas9 nucleus.
On the other hand, the genome editing process of these innovative
technology imposed broad similarity when we are dealing with typographical
 errors in word processor. Due to its provided operational capability, where
one can alter, insert, and replace sequence like elimination and deleting
typos in a document.
The underlying mechanism of the Cas9 nucleus was its capability to
detect the specific sequence of defected double stranded plasmid needed to
be replace with the guided RNA, Generally, it implies that this CRISPR Cas9
system loads similarly into a software or a program that runs its function into
detecting DNA sequence either existing body cells or viral DNA of foreign
invaders in order to develop its appropriate and adaptive immune response.
Prolong, carry out into the process of cutting and altering precise DNA
sequence with the CRISPR Cas9 system guided RNA.
Source: TED TALK: How CRISPR lets us edit our DNA by Jennifer Doudna from
https://www.youtube.com/watch?v=TdBAHexVYzc&t=405s

BONUS: Which graph in the research journal tells the whole story of
their research? (Hint: It is the "smoking-gun" of their research :D)
Meaning, without that data, every experiment they have done is
useless. It is the most important experiment they have made to
answer their research problem/question while the rest are just
supporting details.
A PROGRAMMABLE DUAL-RNA–GUIDED
DNA ENDONUCLEASE IN ADAPTIVE
BACTERIAL IMMUNITY
By Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna,
and Emmanuelle Charpentier

INSIGHTS:

CRISPR Cas9 system had provoke the public’s view on life due to its game
changing gene editing technique brought by two female scientists, Jennifer Doudna and
Emmanuell Charpentier. These prodigies were able to transcend the future of genetics
and its eminence to humanity, taking all of us into closer and clearer vision of what really
happens inside these tiny molecules that make up in every living organism. Research
article by Jinek et al. (2012) presented a profound detail on what is this phenomenal
CRISPR Cas9 System and how does it work to completely achieved the goal of an
accurate gene modification within living cells. Prior to the centralized goals of the pioneers
who introduced the technology, this study reveals the underlying process and molecular
key players, the Cas9 endonuclease founded with two essential RNA, the matured crRNA
and the tracrRNA takes part into this exceptional gene editing process.
This research article argued the Type II system, inhibits the chief ingredient, Cas9
protein was the vital feature of this process generated through the complex formation of
these relative crRNAs. It was stated from the article that the cleavage process occurs into
the interlinked DNA strands was effectuated by the dominant functionalities of the two
guided RNAs. Particularly, the abovementioned claims were strengthened by performing
trials of comparison between existing variables of RNA as independent entity as well as
depending points to one another. The results ascertained the necessary role of the trans
activating tracrRNA for the execution of the Cas9 function to cleave the strands of DNA
as well as triggering the pre-crRNA to operationally founded by the enzyme RNase III.
Moreover, it proved the independence of the standalone presence of the matured crRNA
itself from its primitive mechanism was unreliable to support the Cas9 catalyzing process
in order to form plasmid cleavage into the DNA sequence. That is to say that both tracrNA
and the matured cRNA complementary functions together whereas it gradually changes
the response of Cas9 protein rather than matured crRNA standalone. As they performed
their hand in hand, the envision occurrence of the cleavage process was precisely carried
out to the plasmid yet, it also offered accurate clipping process generating short linear
dsDNA which implies the high potentiality to performed a target binding DNA sequencing.
From the results of the trial, they were able to recognized distinct sequencing of an
upstream base pairs on the DNA strands known as the PAM sequence.
 Thus, this was remarkably showcase through the given figures as the visual
representation of the results of the experimentation which enable the researchers to
thoroughly established a genuine understanding on the genome editing process
performed by the CRISPR Cas9 system. Mainly, Figure 1 was notably the backbone
representation of these modern genome editing tool, CRISPR Cas9 system. Wherein it
exceptionally describes the working mechanism of this technology specifically visually
supported the previously mentioned claims prior to this the discovery. From the mere
visual representation, researchers were able to determine the recipes of success of these
emerging genetic process in particular the commendable RNA molecules as its hallmark
of Cas9 technique. This innovative technological revolution creates avenue for humanity
to deeply grasp into the internal cycle which sustains life of organisms. Supported by
various studies, how the CRISPR technology heralds a promising future for medicine due
to theories that indicates its application as a channel for successful human disease
treatment. Leading into the bigger question, does paradigm in gene modification holds
the future of humanitarian existence giving the assurance of temporarily delaying every
living organism ending scene, simply death and decomposition?

Source: Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012).

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial

Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829
References:

Abbott, T. R., Dhamdhere, G., Liu, Y., Lin, X., Goudy, L., Zeng, L., Chemparathy,
A., Chmura, S., Heaton, N. S., Debs, R., Pande, T., Endy, D., La Russa, M.
F., Lewis, D. B., & Qi, L. S. (2020). Development of CRISPR as an Antiviral
Strategy to Combat SARS-CoV-2 and Influenza. Cell, 181(4), 865–
876.e12. https://doi.org/10.1016/j.cell.2020.04.020

Arber, W., & Linn, S. (1969). DNA modification and restriction. Annual review of
biochemistry, 38, 467–500. PubMed. Retrieved from
https://doi.org/10.1146/annurev.bi.38.070169.002343

Britannica, T. Editors of Encyclopaedia (2020, May 18). Restriction enzyme.

Encyclopedia Britannica. https://www.britannica.com/science/restriction-
enzyme

Centre for Genetics Education. (2020, July). AN INTRODUCTION TO DNA,

GENES AND CHROMOSOMES. Centre for Genetics Education. Retrieved
from https://www.genetics.edu.au/publications-and-resources/facts-
sheets/fact-sheet-1-an-introduction-to-dna-genes-and-chromosomes

Cong L., Zhang F. (2015) Genome Engineering Using CRISPR-Cas9 System.

In: Pruett-Miller S. (eds) Chromosomal Mutagenesis. Methods in
Molecular Biology (Methods and Protocols), vol 1239. Humana Press,
New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_10

Cross, R. (2021, January 25). 8 tools that helped us tackle the coronavirus.
Chemical & Engineering News. Retrieved
from https://cen.acs.org/biological-chemistry/infectious-disease/8-tools-
that-helped-us-tackle-the-coronavirus/99/i3

Cyranoski, D. CRISPR gene-editing tested in a person for the first

time. Nature 539, 479 (2016). Retrieved from
https://doi.org/10.1038/nature.2016.20988
Danna, K., & Nathans, D. (1971). Specific Cleavage of Simian Virus 40 DNA by
Restriction Endonuclease of Hemophilus Influenzae. Proceedings of the
National Academy of Sciences of the United States of America, 68(12), 2913-
2917. Retrieved from http://www.jstor.org/stable/61110

Ding, Q. et al. A TALEN genome-editing system for generating human stem cell-
based disease models. Cell Stem Cell 12, 238–251 (2013). Retrieved from
https://pubmed.ncbi.nlm.nih.gov/23246482/

Ding, R., Long, J., Yuan, M., Jin, Y., Yang, H., Chen, M., Chen, S., & Duan, G.
(2021, April). CRISPR/Cas system: A potential technology for the
prevention and control of COVID-19 and emerging infectious diseases.
Frontiers. Retrieved
from https://www.frontiersin.org/articles/10.3389/fcimb.2021.639108/full#
B1

Doudna, J. (2015, November). How CRISPR lets us edit our DNA [Video].
YouTube. Retrieved
from https://www.youtube.com/watch?v=TdBAHexVYzc&t=405s

Fernández, C. R. (2020, January 14). Seven diseases that CRISPR technology

could cure. Labiotech.eu. Retrieved from https://www.labiotech.eu/best-
biotech/crispr-technology-cure-disease/

Further methods - Genome editing. (n.d.). Startseite - Max-Planck-

Gesellschaft. Retrieved from https://www.mpg.de/11825120/crispr-cas9-
methods

Horvath, P., Romero, D. A., Coûté-Monvoisin, A. C., Richards, M., Deveau, H.,
Moineau, S., Boyaval, P., Fremaux, C., & Barrangou, R. (2008). Diversity,
activity, and evolution of CRISPR loci in Streptococcus
thermophilus. Journal of bacteriology, 190(4), 1401–1412.
10.1128/JB.01415-07. PubMed. Retrieved from
https://pubmed.ncbi.nlm.nih.gov/18065539/

Isaacson, W. (2021, January 11). How mRNA technology could upend the drug
industry. Time. Retrieved from https://time.com/5927342/mrna-covid-
vaccine/
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E.

(2012). A Programmable Dual-RNA-Guided DNA Endonuclease in

Adaptive Bacterial Immunity. Science, 337(6096), 816–821.
https://doi.org/10.1126/science.1225829

Kaiser, J. (2021, June 26). CRISPR injected into the blood treats a genetic
disease for first time. Science AAAS. Retrieved
from https://www.sciencemag.org/news/2021/06/crispr-injected-blood-
treats-genetic-disease-first-time

Lander, E. (2016, January 14). The Heroes of CRISPR. Cell Press

Journal. Retrieved from https://www.cell.com/fulltext/S0092-
8674(15)01705-5#

Ledford, H., & Callaway, E. (2020, October). Pioneers of revolutionary CRISPR

gene editing win chemistry Nobel. Nature. Retrieved from
https://www.nature.com/articles/d41586-020-02765-9

MacDonald, J. (2016, January 21). The revolution will be Geneticized: From

restriction enzymes to CRISPR. JSTOR Daily. Retrieved
from https://daily.jstor.org/revolution-will-geneticized-restriction-enzymes-
crispr/

Marson, A. (2020, October 19). CRISPR-based DNA vaccine enhancer for

COVID-19. Innovative Genomics Institute (IGI). Retrieved
from https://innovativegenomics.org/projects/crispr-based-dna-vaccine-
enhancer-covid-19/

Meselson, M., & Yuan, R., (1968) DNA restriction enzyme from E. coli. Nature 217,
1110–1114. Retrieved from doi:10.1038/2171110a0

National Geographic Society. (2019, May 23). History of the cell: Discovering the
cell. Retrieved from https://www.nationalgeographic.org/article/history-cell-
discovering-cell/
Pray, L. A. (2008). Restriction enzymes. Nature Education. Retrieved from
https://www.nature.com/scitable/topicpage/restriction-enzymes-545/

Ran, F., Hsu, P., Wright, J. et al. (2013) Genome engineering using the CRISPR-
Cas9 system. Nature Protocols 8, 2281–2308. 10.1038/nprot.2013.143.
Retrieved from https://www.nature.com/articles/nprot.2013.143

Roberts, R. J. (2005, April 26). How restriction enzymes became the workhorses
of molecular biology. PNAS. Retrieved from
https://www.pnas.org/content/102/17/5905