Journal Review on Gene-Knock-out

CRISPR/Cas 9: A Cure for Cancer and other Genetic Diseases

Judy Ann G. Advincula

Jean Rose E. Genova
Charmaine S. Nemiz

Background

Several therapies for cancers have been evolving such as chemotherapy, radiotherapy and
surgery or even combination of those which were made available by technologies for the
betterment of projection in patients to reduce the cancerous cells and somehow increase the
lifespan of the patient to a maximum of five years, however harmful side effects and toxicity can be
associated with these treatment that can increase the mortality reduce the quality of life.

Therefore this research article presented alternative source of treatment for cancer and
other genetic diseases by the use of genome edition technology. The journal reviewed is about gene
knock out, a kind of gene targeting technology that is used to modify genomes of any living
organisms or when a mutation inactivates a gene function (MyBioSource, 2007). Specifically the
journal tackled the use of clustered regularly interspaced short palindromic repeats (CRISPR)
associated with Cas9 as a cure for cancer and other genetic diseases.

Previously, several therapies to treat cancer were introduced but none sustained for long
time. Major causes of failure include the development of the self-resistance and the deleterious side
effects. Previously DNA domain binding proteins zinc fingers nucleases (ZFNs) and transcription
activator-like effector nucleases (TALENs) were employed to treat cancers but their efficiency was
limited due to their inability to effectively target the epigenetic changes that occur during
carcinogenesis. Recently a more versatile genome editing technology, clustered regularly
interspaced short palindromic sequences (CRISPRs) associated with HNH domain protein Cas9
shows promise towards reliable long term cancer therapy. CRISPR/Cas9 is an adaptive immune
system in bacteria and archaea against phage invasion in natural environment. Bacteria evolve this
system through capturing DNA sequences and used it as a memory to be identified as enemy and
destroy it on its attack in future.
 This natural adaptive immunity of bacteria and archaea can be redesigned to achieve
desired genome editing and more importantly repurpose it as a therapy against long awaiting
cancerous and genetic disorders. Epigenetic changes defines the environment for cancer
development. The mutations in tissue development genes stimulate cancer development.

The present review is an attempt to see the applicability of CRISPR/Cas9 system in cancer
and genetic disease therapies. Furthermore, several genetic mutations and suitability to
CRISPR/Cas9 system is explored to provide researchers to focus on the translation of laboratory
research to clinics.

Methods

In this study the application CRIPRS/Cas 9 was done through studying the
mechanism of this genome editing technology, identifying the carriers of CRISPR/Cas9 to
the living cells, differentiating two DNA repair pathways and presenting challenges in
CRISPR/Cas9 clinical therapeutics.

Describe the Challenges in

mechanism of genome CRISPR/Cas9 Clinical
editing technology Therapeutics

Identify the carriers of Differentiate the two

CRISPR/Cas9 DNA repair pathways

The paper presented the mechanism of genome editing technology especially in the new
generation sequencing. The NGS technologies include whole genome sequencing, whole exome
sequencing, RNA sequencing, reduced representation bisulfite sequencing, and chromatin
immunoprecipitation sequencing each of which is employed for specific objectives. In cancers often
the whole exome sequencing is performed to get specific mutations at the cellular levels. The design
of any therapeutic technology at molecular level that can cure diseases should have the ability to
precisely correct malfunctioned cells and pathways.
 Furthermore, the paper stated that the difficulties in uniform and sustained transportation
of CRISPR system to cells was needed to be addressed in order to secure the success of the
CRISPR/Cas9 system in curing cancer and other diseases.

Moreover, since the gene editing ability of Cas9 is of paramount importance, the journal
presented the two natural pathways present in the cell of DNA repair was discussed; non-
homologous end joining (NHEJ) and homology directed repair (HDR) to ensure the specificity and
accuracy of the gene editing process.

On the other hand, since CRISPR-Cas9 genome editing technology holds promise to
personalized medicine, human genetic modification and the development of new drugs challenges
and ethical concerns on the use of CRISPRCas9 genome editing technology was raised. On the latter
part of this paper a temporary moratorium was cited in order for the technology to allow the
scientific community and other stakeholders to engage in a broad-based discussion to map the way
forward for this technology.

Results and Discussion

Based on the review of the paper, the following were the results of the study:

A. Mechanism of Genome-Editing Technology

CRISPR-Cas9 found its origin from type II CRISPR-Cas systems, which enable the bacteria to
mount an adaptive immunity against invading viruses and plasmids. When viruses or plasmids
enter into a bacterial cell, CRISPR system allows the integration of the short viral DNA molecules
into the CRISPR locus. The biogenesis of CRISPR RNA involves the transcription of CRISPR sequence
into RNA, which is subsequently used with proteins encoded by Cas genes to form interference
complexes that are used in the formation of RNA molecules to base pair with matching sequences of
viral DNA. The CRISPR sequence CRISPRs, “clustered regularly interspaced short palindromic
repeats”, are short DNA repeats of viral origin found in the bacterial genome. Cas (CRISPR-
associated) is an endonuclease that recognizes and cut the DNA. CRISPR-Cas complex recognizes
the DNA of the invading virus and guides the Cas protein to cleave the virus.

B. Carriers of CRISPR/Cas9 to the Cell

The CRISPR/Cas 9 system has taken the world of gene editing by storm, it has become
resort technique when it comes to mutating and editing DNA in bioscience research. It only requires
two component, the cas 9 enzymes to s nit target DNA and the RNA molecule called the single
strand guide RNA that directs the cas 9 enzyme to the precise spot for complementary DNA pairing.
The delivery efficiency of the Cas9 endonucleus along with the tRNA directly affects the success of
genome editing. There are several method to express these components in living cells, the non-viral
based gene delivery (lipid mediated transfection or Calcium-phosphate transfection,
electroporation) or viral based gene delivery (use of Lentivirus, Adenovirus and Adeno-Associated
Virus). Also, the methods involve electroporation microinjection, lipofection nucleofection. The
main disadvantage of using plasmid is the random integration of plasmid or its part in both off-
target and on-target sites leading to insertion inactivation of genes. To address these shortcomings,
several efforts were put in to deliver Cas9 protein in conjugation with cell penetrating proteins
(CPPs) complexed with guide RNA (gRNA) that forms nanoparticles with positive charge showed
efficient disruption of genes. An enhanced delivery vehicle inspired from DNA nanoclews and DNA
nanoparticles is recently been reported having the capability of simultaneously delivering the Cas9
protein and sgRNA to human cell nuclei and disrupt genes efficiently while maintaining cell
viability. Viral vectors such as Adeno-associated virus (AAV), lenti-virus are broadly used as gene
delivering tool due to efficient introduction of an exogenous DNA fragment into genome by robust
HDR. It is of paramount importance to develop tools and methods to efficiently and specifically
stimulating or inhibiting gene expression to achieve desired results for cancer therapy.

C. Differentiate the two DNA repair pathways

The dsDNA break by Cas9 is followed by two natural pathways present in the cell; Non-
homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ is naturally favored
pathway for gene correction in nature but is error prone and causes undesired mutations hence is
not suitable for the application of CRISPR as therapeutic agents. HDR on the other hand is accurate
and error free but is not naturally favored pathway for DSBs correction, hence requires means to
make HDR to be favored over NHEJ in natural environment to efficiently translate CRISPRs benefits
to clinics.

Furthermore, there are some obstacles that limits the commercial therapeutic application of
CRISPR. Some of the challenges faced researchers on this paper was the specificity of the CRISPRs in
which many clinical laboratories are concerned about its off-target effects and the ways that can
minimize those off-targets and develop clinical assays to measure. Several advances are made in
delivering nucleases to destination cells ex vivo and in vivo but there is still need to improve the
delivery systems for realizing the dream of CRISPRs therapeutics. Also aside from the specificity,
another hinder to its success is the isolation of mutant cells (having DNA of interest) from a diverse
population of cells.

Summary

Genome editing technologies may in the future have therapeutic potential for various
incurable diseases: cancer, genetic disorders. Genome editing of somatic cells, on its various clinical
stages, is a promising area of therapeutic development in which one of these is the use of
CRISPR/Cas9 technology. In defining its superior use, researchers followed the recent advances
that have been made in producing CRISPR/Cas9 as a therapy of choice. Researchers have also
provided important genetic mutations where CRISPRs can be repurposed to create adaptive
immunity to fight and edit genetic mutations causing it. However, challenges to CRISPR technology
with emphasis on ability of pathogens that may evolve against CRISPRs need to be addressed.

Reference

Khan, F.A., Pandupuspitasari, N.S., Chun-Jie H., Ao, Z.,Jamal, M., Zohaib, A., Hakim, M.R., Shujun,
Z.,(2016).CRISPR/Cas9: A cure for cancer and other genetic diseases. Retrieved from
www.impactjournals.com/oncotarget.