Advances in Cancer Biology - Metastasis 3 (2021) 100012

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Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Epigenetic targeting for lung cancer treatment via CRISPR/Cas9 technology

Ocean Khajuria *, 1, Neha Sharma 1
Shri Mata Vaishno Devi University, School of Biotechnology, Katra, 182320, India

A R T I C L E I N F O A B S T R A C T

Keywords: The growth of CRISPR/Cas9 system and findings of various Cas9 protein forms along with its improved delivery
Oncogene methodologies has enhanced the practical applications of gene editing. Inducing desired gene of interest to
dCas9 correcting disease related mutations and to suppress or activate a gene of interest efficiently is now practically
Lung cancer
possible with the help CRISPR/Cas9 system. The main cause of lung cancer is the dysregulation of epigenetic
Tumor suppressor genes
CRISPR/Cas9
markers which bring transformation in normal cells. Targeting epigenetic modification or epigenetic regulatory
enzymes with the help of CRISPR/Cas9 system might be a vital step for cancer therapy.

1. Introduction of the DNA site in the CRISPR-Cas9 system is controlled by RNA–DNA

The most frequent type of cancer affecting humans is lung cancer that
accounts for 2.09 million diagnosis together with 1.76 million deaths
globally in 2018 [1]. Around 85% of the lung cancer is non-small cell
lung cancer (NSCLC) having five year of overall survival rate whereas
15–20% lung cancer are small cell lung cancer (SCLC). The treatment of
NSCLC is determined by the cancer stage, which is categorised as 0, I, II,
IIIA, IIIB, IVA, IVB using the TNM (Tumor Node Metastasis) classifica-
tion. Usually the condition is diagnosed at advance stage wherein prog-
nosis remains terrible with limited therapeutic alternatives [2].
Certain therapeutic strategies for lung cancer patients encompass
surgery, radiation therapy, chemotherapy, targeted therapy, and immu-
notherapy. The enhanced tolerance of DNA damage, increased efflux of
drug, decreased permeability and enzymatic deactivation allow cancer
cells to survive defying all the treatments in numerous cases. Enhancing
therapeutic techniques to ensure patients survival has become prime
importance [3].
The main contribution of this work is to highlight the importance and
pre-eminence of CRISPR/Cas 9 as genome editing tool that has paved its
way for advancement in lung cancer treatment. CRISPR-Cas is a genome
memory system that occurs naturally in diverse bacteria and archaea.
When present in a natural host, CRISPR-Cas confers selective immune
resistance against foreign genetic material of viral or plasmid origin [4].
The CRISPR/Cas system has a number of advantages over other two
established genome editing tools. The core difference is the recognition
interactions. This offers numerous possible circumstances over
zinx-finger nucleases(ZFNs) and transcription activator-like effector nu-
cleases (TALENs), including simple structure for any genomic targets,
simple prediction with respect to off target sites, and the chance of
changing a few genomic locales simultaneously (multiplexing) [5].
This study helped us in comprehending epigenetics as significant
subject of cancer research. Collective proof demonstrated that malig-
nant change of typical cells and tumor maintenance requires broad
epigenomics [6].We also discussed a few possibilities of CRISPR/Cas9
as a potential method for targeting cancer epigenome for disease
treatment.
2. Risk factors
2.1. Tobacco smoking
Smoking is the major cause of lung cancer in all histological types [7].
Since the mid-1960s, epidemiological studies have shown that cigarette
smoke has a carcinogenic impact on the lungs, which has been recog-
nized by public health and regulatory authorities [8].
Nicotine, a natural alkaloid that acts as an acetylcholine agonist and
binds to nicotinic acetylcholine receptors (nAChR) in the nervous system,
induces the release of neurotransmitters such as dopamine, serotonin,
norepinephrine, endorphins, and gamma-amino butyric acid into the
blood stream [9]. Tobacco combustion produces at least 60 known

* Corresponding author.
E-mail addresses: oceansharma2617@gmail.com (O. Khajuria), 24sharma.n@gmail.com (N. Sharma).
1
All the authors contributed equally to this work.

https://doi.org/10.1016/j.adcanc.2021.100012
Received 20 July 2021; Received in revised form 28 September 2021; Accepted 4 October 2021
Available online 7 October 2021
2667-3940/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
O. Khajuria, N. Sharma
carcinogens. PAH (polycyclic aromatic hydrocarbons) being the most
expressive [10].
2.2. Genetics
A positive family history of lung cancer has been related to a 1.7-fold
increased risk of developing lung cancer. The risk of lung cancer is
increased 2 to 4 times in first-degree relatives of lung cancer patients,
even though personal smoking history is taken into account [11]. Ac-
cording to Genome wide association studies, chromosome regions 5p15,
15q25-26 and 6q21 are associated with increased risk for lung cancer
[12].
2.3. Never smokers/non-smokers
According to global estimates, 15% of lung cancers in men and up to
53% in women are not caused by smoking, indicating a clear gender bias
among never smokers. Furthermore, never smokers accounts up to 25%
of all lung cancer cases worldwide [13]. If never smoker's lung cancer is
counted separately, it would be the seventh leading cause of cancer death
worldwide, followed by cervical, pancreatic, and prostate cancers [14].
According to a study in the United States, never smokers account for 19%
of lung cancer in women and 9% of lung cancer in men [15]. Up to 80%
of women with lung cancer in South Asian countries have never smoked.
From 1990 to 1995 to 2011 to 2013, the proportion of never smokers
with adenocarcinoma non-small cell lung cancer rose from 8.0% to
14.9% [16]. With the declining smoking prevalence and the increased
number of lung cancer among nonsmokers, there is a greater need to
better understand other etiologic factors that contribute to lung cancer
besides tobacco use.
2.4. Epigenetics
The epigenetic mechanisms which influence the gene expression
regulation to a vast extent are DNA methylation, histone modifications
and miRNAs [13]. Since their prominent role in gene expression they
control important roles like normal cell differentiation, proliferation and
its function [17]. Amassing of genetic and epigenetic events in the res-
piratory epithelium induce lung cancer. In spite of the fact that mutations
and alternations in copy number consume a significant part in onco-
genesis, the epigenetic modifications stand more recurrent than somatic
mutations in lung cancer [18]. Epigenetic dysregulation prompts
numerous genetic instability, bringing about the gain of hereditary
changes in tumor-suppressor genes and accordingly enacting hereditary
mutations in oncogenes [19].
3. Epigenetic mechanism
3.1. DNA methylation
The inactivation of tumor suppression genes (TSGs) via promoter
methylation also known as hypermethylation is a hallmark of lung cancer
which occurs in early stages of carcinogenesis [20]. Methylation of
promoter APC, RASSF1A, ESR1, HOXC9 etc is linked with NSCLC stage 1
[21]. Methylation at CpG sites can likewise prompt point changes via
deamination at the 5- methylcytosine (5-meC) or elevation of
cancer-causing agents. The hydrolytic deamination of 5-mec leads to C-T
transition [22]. It has been recently identified in 2019 by Verena
Deutschmeyer et al. that a potential tumor suppressor zygote arrest 1
(ZAR1) by epigenetic inactivation influences the lung carcinogenesis
[23].
3.2. Histone modification
Notwithstanding, similar to DNA methylation, histone methylation is
likewise reversible. Demethylases helps in removal of methyl groups
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Advances in Cancer Biology - Metastasis 3 (2021) 100012
from histone amino acid residues. Acetylation of histone tails has been
emphatically connected to carcinogenesis [24]. Histone deacetyla-
se(HDAC) overexpression leads to repression of tumor suppressor genes.
Sasaki et al. [25] detailed that the size of the lung tumor is associated
with the expression of HDAC1. Whereas Bartling et al. discovered that
HDAC3 expression was enhanced in lung squamous cancer cells [26].
HDAC inhibitors have been evolved as a new anti-cancer agents owing to
their ability to induce apoptosis, cell cycle arrest, autophagy, suppressing
tumor angiogenesis and many other mechanisms [27].
3.3. miRNAs
Certain groups of 24 miRNAs were suggestively related to lung cancer
development. It was observed that there was remarkable increase in 6
miRNA (miR-146b-3p, mi-566, miR-550 etc) whereas diminishing of
other two microRNAs (miR-339–5p along with miR-656) was reported in
the sample [28]. The uprise of miRNAs was predominantly seen in the
early stages of disease, revealing their role in timely diagnosis [23].
4. Action of Cas9 nuclease on cancer epigenetics
Jinek et al. created sgRNA for S. pyogenes in which crRNA and
tracrRNA complements with the type II system. The system developed
was equipped with DNA binding ability and cleavage property of the
complementary DNA, target as coordinated by sgRNA, by passing the
processing part [29]. A lentiviral construct having dCas9 or a Cas9
variation, four sgRNAs, each of those from independent Pol III promoters
and a reporter gene, was developed significantly for human cells. This
framework was effective for encouraging multiplex quality editing. It's
not just restricted to targeting multiple genes; the productivity of guide
RNA is to identify single nucleotide polymorphism additionally em-
powers allele specific gene adjustment [30]. Therefore, designing
CRISPR/Cas9-based mechanisms for targeting cancerous epigenetic
regulators in a precise manner is more efficient. Nickase Cas9 was
additionally investigated to refine the target specificity of Cas9. Joined
with two corresponding counterbalance sgRNAs, it was observed that
Cas9 was able to produce indels, consequently decreasing undesirable
cleavage since the base excision repair mechanism fixes single stranded
off-target nicks individually with high fidelity [31].
dCas9 along with effector helps in repression or activation of gene
without modifying DNA. At the point when intertwined to transcriptional
actuation, VP64 and VP64-p65-Rta fusion domains, dCas9 effector pro-
teins initiate expression of gene at various levels relying upon the quality
of activator utilized and the general dCas9 binding site to start site of the
transcription [26]. The dCas9-fusion construct fit for transcriptional
tuning vary in utilization of transcriptional modular (TM). A portion of
TMs are activators while others have inhibitory role. Krüppel-related box
(KRAB) area is generally utilized for inhibition of transcription while
herpes simplex viral protein 16 (VP16) oligomers are usually utilized the
activation of transcription [32]. Studies have shown that, epigenetic
factors like, ten eleven translocations (TET) protein families, generally
work as tumor silencers, were oftentimes gets inactivated due to epige-
netic changes, while others including, LSD1, NSD2 and EZH2 have ca-
pacities as tumor drivers and were usually found overexpressed by
epigenetic or hereditary systems [33]. Thus, it may be successful idea to
produce transcriptional regulators based on CRISPR/Cas9 system that
eventually assists with re-establishing or suppressing the expression of
such enzymes related to cancers.
5. Targets for CRISPR/Cas9
5.1. Tumor-suppressor gene
As depicted in Fig. 1, Tumor suppressor genes [TSGs] linked with lung
cancer comprises RB, TP53, MCC, APH, NM23 and APC [34]. The
expression of TSGs can restrain the proliferation of cells, encourage cell
O. Khajuria, N. Sharma
Fig. 1. The action Cas9 nuclease could either lead to knockouts or repair of genes which would inactivate and activate oncogenes and TSG's respectively. As a result it
will induce anti lung cancer effects.
differentiation, hinder migration of cells, and adversely regulates tumor
progression [35] Loss of function, knockout, or mutation of TSGs brings
about the initiation of oncogenes, prompting tumor genesis. TSGs can be
easily targeted by CRISPR/Cas9 tool for treatment. With the help of
CRISPR technology these genes can be proficiently targeted for mending
and the ability of tumor repression can be re-established. Many TSGs are
currently being studied using CRISPR/Cas9 technology [30].
5.2. Oncogenes
Some of the proto-oncogenes related to lung cancer include MYC,
RAS, ERBB1, ERBB2, ROS1, among others [36]. It has been seen that RAS
gene mutations was found in 30% of NSCLC and overexpression of
c-ERBB-1 protein in 75% of lung adenocarcinomas and 95% of lung
squamous cell carcinomas [37]. Large numbers of oncogenes have been
Fig. 2. This figure represents the different routes of Cas9 delivery. 1–2 are physical methods of delivery and 3–5 are chemical methods of delivery system and Cas9 can
possibly be administered as mRNAs, proteins and DNA molecule.
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Advances in Cancer Biology - Metastasis 3 (2021) 100012
identified as a candidate targets for cancer treatment. Epidermal growth
factor receptor (EGFR), focal adhesion kinase (FAK), neuroectoderm
stem cell marker (NESTIN), remodelling and spacer factor (RSF1),
(CTNND2) and insulin-like growth factor 1 receptor (IGF1R) have been
examined in recent years for the treatment of lung cancer via CRISPR/-
Cas9 gene editing [38]. Using the oncogenic mutant EGFR allele
CRISPR/Cas9 knockout inhibits the growth and proliferation of H1975,
A549, and H1650 lung cancer cell lines, and decrease tumor volumes in
H1975 or A549 cell implanted xenograft mice, EGFR mutation-specific
Cas9 decreased tumor size by 81.5% and 78.3% compared to that of
phosphate buffered saline (PBS) - and Ad/Cas9-treated controls [33]. In-
vitro experiments on different cancer cell lines have revealed that the
editing method based on CRISPR/Cas9 was a better approach for tar-
geting oncogenic KRAS mutation at codon-12 that was associated with
the proliferation of cancer cells [39].
O. Khajuria, N. Sharma
6. Delivery system for CRISPR-Cas9
Cas9 can possibly be administered as mRNAs, proteins and DNA
molecule as depicted in Fig. 2. Plasmid DNA is comparatively stable and
less expensive. For DNA-encoded delivery to be functional and success-
ful, the system should breach both the barriers of cellular and nuclear
membrane [34]. For the active expression of Cas9, great care is taken
with this type of format with the promoter sequence or the replication
sequence encrypted in the plasmid. An alternative approach could be to
encode it as an mRNA making it easier to express in cytoplasm and thus
by passing the difficult task of crossing the nuclear membrane. For
long-term gene therapy purposes, mRNAs are not sufficiently stable.
Nevertheless, even transient articulation and ability will leave the he-
reditary change perpetual for the activity of Cas9 nuclease, which is the
reason why Cas9 mRNA is commonly used, for example, Drosophila,
zebrafish, Xenopus and mouse, in both cell culture and model creatures
[40,41].
In its native protein type, is the third choice to deliver Cas9. Due to
enormous size and charge of protein molecule, it is always considered to
be the most problematic delivery design, but Cas9 protein delivery has
recently found to be successful in both in vitro and in vivo systems [ 36].
A vehicle is necessary for the delivery of payload to the target cells,
such vehicles are called delivery vehicles or vectors. Vectors can be
categorised in viral and non-viral vectors. The most popular method for
the expression of the Cas9 is the use of viruses. To extract the repro-
ductive genes responsible for replication, viral particles have been
designed to replace them with a therapeutic transgene of interest [42].
The Adeno associated virus (AAV) vector is the most common. For the
transmission of cas9 in the ex vivo setting, adenovirus and lentivirus have
already been used and achieved highly efficient gene disruption [43].
There are various non-viral Cas9 transfer methods (whether as DNA,
mRNA or protein). Microscopic pipettes have made it possible to move
diverse macromolecules through microinjection, including CRISPR
components [38]. Microinjections may deliver cargo either in the cyto-
plasm or, in fertilised egg pronucleus in some cases. High efficiency,
managed measurement, and an assurance that the load is delivered
precisely to the proposed site are given by microinjection. Recently, in
human embryo microinjection-based repair of point mutation via Cas9
was successfully reported [44].
For CRISPR DNA, mRNA, or RNPs (ribonucleoprotein), lipid-based
carriers are a common and effective delivery method. Lipid nano-
particles were spontaneously generated in an aqueous solution to shield
the hydrophobic tail from the solvent [45]. Payload can be integrated
inside a lipid nanoparticle or compounded with a lipid nanoparticle
through easy mixing [40]. The lipid carrier protects the payload from the
host's enzymatic degradation or immune response. Lipofectamine is a
best example of commercial lipid transfection delivery vehicle that is
used for delivery of nucleic acids and protein editing genes [46]. Delivery
of Cas9 RNP by the using nucleic acid has also recently been reported
[47]. Where there is no need for the encapsulation of proteins and nucleic
acids as they can be modified chemically to achieve an improved de-
livery. Use of cell-penetrating peptides (CPPs) is one common approach
to this [48]. Cas9 protein and gRNA conjugation with CPPs may serve as
a way of improving the delivery of protein. But it does not overcome the
protease degradation issue that can be done by encapsulation.
Funding
This work is not supported by any means of funding.
Future prospects
CRISPR/Cas9 system has emerged to be one of the most proficient
tools for gene editing purposes in past few years. Larger size, off target
effects and ability to target only PAM sequences are some aspects of this
system which requires utmost attention and solution. Still it must be
4
Advances in Cancer Biology - Metastasis 3 (2021) 100012
refined as a technique. Along with its far-fetched efficacy it holds the
potential for improving treatment processes of diseases. Rapid
advancement of this technique can help in improving the competence of
various treatments in future. CRISPR/Cas9 is effectively being used for
gene editing purposes in lung cancer treatment research and with its
advancements in future it can become the sole reliable tool for the
treatment purpose.
Declaration of competing interest
We wish to confirm that there are no known conflicts of interest
associated with the publication and there has no significant financial
support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all
named authors and corresponding author will be the sole contact for
Editorial process. We further confirm that we have provided a current,
correct email address which is accessible by the corresponding author.
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