Adopting CRISPR-mediated Genomic Editing Technique on the

Treatment of Lung Cancer: Using Revolutionary Genomic Editing

Technique to Treat Serious Human Disease

Sixian Chen

Beijing Huijia Private School, Beijing China

Keywords: CRISPR, Lung Cancer, EGFR Gene, EML4-ALK Fusion Gene, TSLC1, P107 and P130.

Abstract: CRISPR system was first discovered in E. coli in 1987 by Japanese scientist Yoshizumi Ishino and his team.

Since its emergence, it has been widely applied for a variety of medical use, including lung cancer. Traditional

treatments of lung cancer gradually lost in their effectiveness by inducing drug resitance. Therefore, more

influencing and innovative technologies are needed urgently, and CRISPR-mediated genomic editing

technique is one of them. CRISPR system helps scientist to construct tumor model, to identify certain lung

cancer related genes as well as deleting or repairing those cancer genes which can be done by targeting specific

genes and either inhibit or activate its function. The applications of CRISPR system are now developed in a

flying speed that it already moves to the clinical testing stage. Ideally, within a decade, the adoption of

CRISPR system on treating lung cancer can be popularized widely. This review summarizes the

characteristics of lung cancer and the application of CRISPR system on treating lung cancer. Potential target

genes related with lung cancer will be discussed including EFGR, EML4 fusion gene, TSLC1 etc. The

application of CRISPR system on deleting various types of cancer gene will be introduced, too. In order to

generalize CRISPR technology into human cases, more in-depth investigations of the usage of this system are

necessary for future studies.

1 INTRODUCTION

Lung cancer is a common type of cancer happened in

lungs that leads to roughly 25% of death among all

cancer cases. Its morbidity rate is ranked at the top

level among a lot of developing countries including

China, Europe and so on. There’re mainly two types

of lung cancer – non-small cell lung cancer (NSCLC)

and small cell lung cancer (SCLC). NSCLC takes up

approximately 80% - 85% of all lung cancer cases

(National Comprehensive Cancer Network). There’re

a variety of subtypes within NSCLC including

adenocarcinoma, large cell carcinoma, squamous cell

carcinoma and so on. Although each of them damages

different parts of the lungs, they’re collectively

categorized as non-small cell lung cancer since they

possess similar treatment as well as prognoses. On the

other hand, SCLC, or oat cell cancer occupies 10% -

15% lung cancer cases. It generally grows in a more

rapid rate compared with NSCLC. The metastatic

https://orcid.org/0000-0003-4132-7707

speed is also faster in SCLC patients that by the time

the cancer is discovered, it has already diverted to

other organs or tissues. It’s also possible for tumor

cells to be metastasized to the lungs from other parts

of the body, but in this case, the tumor will be named

after the primary cancer site instead of lung cancer

(Niederhuber et al. 2020).

Due to its low surviving rate, the treatment of lung

cancer is always the focus of clinical research. The

pathology of lung cancer is mostly due to the

mutation of certain genes. This mainly includes the

transformation of proto-oncogene into oncogene and

the inactivation of tumor suppressor gene. For the

past decade, people have tried a huge number of

therapeutic pathways to treat lung cancer,

chemotherapy is one of the most common fashion.

However, despite the various types of chemotherapy,

many patients developed drug resistance after

receiving chemotherapy which lower the efficiency

of the treatment significantly. Traditional way of

168

Chen, S.

Adopting CRISPR-mediated Genomic Editing Technique on the Treatment of Lung Cancer: Using Revolutionary Genomic Editing Technique to Treat Serious Human Disease.

DOI: 10.5220/0011228700003444

In Proceedings of the 2nd Conference on Artiﬁcial Intelligence and Healthcare (CAIH 2021), pages 168-174

ISBN: 978-989-758-594-4

therapy is already behind the times. To invent new

creative way of treatment, it’s crucial to understand

the causes, mechanisms, and pathologies of lung

cancer. Among all possible reasons, genetic mutation

is considered as the most essential cause of lung

cancer. There’re numerous types of gene that might

induce lung cancer after being either activated or

inhibited. Considering the significant role of mutated

genes play in lung cancer, scientists start to

investigate cancer gene therapy, a therapy that

specifically designed for editing human genome.

Cancer gene therapy is a broad concept that

include any treatment related with active cancer

genes. In the past few years, using gene editing

technologies to treat lung cancer has taken a lot of

researchers’ attentions. CRISPR system is one of

them. CRISPR system is an efficient tool used to edit

the genome of cells. The essential components of this

system include single-guide RNA (sgRNA) and

nuclease in which sgRNA guide the nuclease to cause

a DNA cleavage at the targeted site. This enables

researchers to edit the removed site, normally by

introducing a DNA repair. There are several other

types of gene editing technologies including zinc-

finger endonucleases (ZFNs), transcription activator-

like effector nuclease (TALENs) and so on.

Compared with them, CRISPR system is more

accurate and straightforward. It also allows

researcher to target or edit any type of gene.

There’s a huge amount of research aimed to

investigate lung cancer and the application of

CRISPR system on treating lung cancer. This review

will focus on discussing the pathology of lung cancer,

the traditional treatment of lung cancer as well as the

application of gene editing technology, specifically

CRISPR system on treating lung cancer.

2 PATHOLOGY OF LUNG

CANCER

The pathogenesis of lung cancer is mostly caused by

the mutation of protooncogenes into oncogenes,

inactivation of tumor suppressor genes, insensitivity

of cancerous treatment as well as dysfunction of

immune system (Fig 1) (Jiang, Lin, & Zhao 2019)

Figure 1: Summary of 4 main pathological causes of lung

cancer.

2.1 Mutations of Protooncogene

Cancers are basically caused by mutation of specific

genes. Gene that works in its normal state is called

protooncogene which is in charge of regulating the

progress of cell division. In cancer, protooncogene

will mutate into oncogene which will significantly

disturb the normal machinery of cell division. This

will lead to an uncontrollable proliferation of cancer

cells. Below shows several common types of

oncogenes related with lung cancer.

2.1.1 Epidermal Growth Factor Receptor

(EGFR)

Epidermal growth factor receptor (EGFR) gene

mutation is associated with 13% cases of lung cancer.

It’s commonly detected in non-smoker or Asian

patients with NSCLC (Soda et al. 2007). EFGR gene

is located at chromosome 7. It is belonged to a class

of receptor tyrosine kinases named HER/erbB. The

homodimerization or heterodimerization of this class

of receptors will induce a tyrosine kinase activity.

This process will start a cascade of reactions which

will eventually couple the receptor to the pathway of

downstream signaling. This signaling will cause

various cancerous phenomenon like decreased

apoptosis (Koivunen et al. 2008). The expression of

EGFR gene is the main inducing factor for rapid cell

proliferation, angiogenesis, oncogenesis, and other

cancerous symptoms. EGFR mutation is mostly

happened when there’s a deletion in exon 19 or a

missense in exon 21 (Fig 2). In the missense mutation

circumstance, one of the thymine molecules is

substituted by a guanine molecule (Koo et al. 2017).

Adopting CRISPR-mediated Genomic Editing Technique on the Treatment of Lung Cancer: Using Revolutionary Genomic Editing

Technique to Treat Serious Human Disease

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Figure 2. Mutation of EGFR gene due to the missense of exon 21. The thymine molecule is replaced by a guanine molecule

which turn the code for amino acid leucine into arginine.

2.1.2 EML4-ALK Fusion Gene

Anaplastic lymphoma kinase (ALK) is a type of

enzyme discovered from chromosomal translocation

that will induce the formation of fusion protein. In

this fusion, COOH-terminal donated by ALK is

combined with a NH2 terminal from other genes.

Recently, it is discovered that the fusion of ALK with

echinoderm microtubule- associated protein-like 4

(EML4) might lead to the onset of lung cancer.

Similarly, both ALK and EML4 are located at

chromosome 2 and are separated by 12Mb (millions

of base pairs). There’re a variety of types of variants

of EML4-ALK (Fig 3). For instance, when exon 20-

29 of ALK is fused with either exon 1-13 or exon 1-

20 of EMLK. The previous one is referred to variant

1 while the latter one is referred to variant 2. Both

of the variants are responsible for initiation and

maintenance of lung cancer. Expression of EML4-

ALK gene was inhibited by injection TAE684 which

is an ALK kinase inhibitor in mice sample. This

causes a cease in the size of lung tumor which further

suggests that lung cancer is related with the mutation

of EML4-ALK gene.

Figure 3: Example of 4 different types of EML4-ALK

fusion gene variants with their mutated site and specific

locations.

2.2 Tumor Suppressor Gene

Tumor suppressor gene is a type of gene that can

prevent the growth of tumor. Inactivation of tumor

suppressor gene will lead to a rapid growth of cancer

cells.

2.2.1 TSLC1

A majority of tumor suppressor activities are located

at a 100-kb segment 11q23.2. Kuramochi et al.

conducted a genetic research by adopting yeast

artificial chromosome (YACs) at chromosome 11

(

Kuramochi et al. 2001)

. The study aimed to localize the

existence of tumor suppressor genes in a small

section at 11q23.2. By transferring the overlapped

YACs region from human and nude mice, researchers

have successfully localized a tumor suppressor gene

at the central 700-kb segment of a 1.6-Mb YAC

which is known as TSLC1. Scientists have found that

the expression of TSLC1 is either reduced or absent

in NSCLC as well as several other types of lung

cancer cell lines. Loss of 11q23.2 which cause the

deletion of one allele of TSLC1 was found in 40%

cases of SCLC. The expression of TSLC1 is even

more inhibited with the promoter methylation in

those cell lines. As expected, by reactivating TSLC1,

there’s a significant suppression of malignant

phenotype in lung cancer cells.

2.2.2 p107 and p130

Ng, S. R. et al. (2020) aimed to use CRISPR system

to model lung cancers that are caused by the mutation

of tumor suppressor gene by targeting and

reactivating tumor suppressor gene that are once

dormant. To do this, they target 2 members of

retinoblastoma protein, p107 and p130. The mutation

of retinoblastoma protein is found to happen in 6% of

SCLC patients. Researcher made sgRNA to target

p107 and p130 genes. The validity of those

sgRNAs

was tested in vitro by Western blot to ensure if there’s

a decrease in retinoblastoma protein level.

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To test whether the system is worked in mice or

not, researcher infect mouses with Ad5-USEC

vectors which will express the sgRNA that target

p107 or p130 gene. Then, they conducted a in vivo

bioluminescence imaging to monitor the

circumstance of lung tumor in animal’s body. After a

period of time, researchers detected an acute level of

luciferase activity in both p107 infected and p130

infected animals which is accordant with the

accelerated tumor progression. The average survival

rate within those mouses have decreases

significantly, too. This verifies the tumor suppressing

function of p107 and p130 gene.

2.3 Insensitivity toward

Chemotherapeutic Drugs

In order to treat cancer, chemotherapy is an effective

and frequently used therapeutic method.

Unfortunately, the resistance of patient’s body toward

chemotherapeutic drugs has increased a lot from the

past few years. In such condition, some of the

previously prevailed market drugs are not as effective

as they were used to be For instance, cisplatin-

containing regimens is a commonly used

chemotherapeutic method to treat lung cancer, while

the sensitivity of this cytotoxic drug to NSCLC

patients still remain indistinct. Morodomi et al.

conducted a research to investigate the sensitivity of

chemotherapeutic drugs toward lung cancer patient

with different types of gene mutation (Morodomi et

al. 2014). The study found that patients with EML4-

ALK fusion gene is less sensitive to cytotoxic

chemotherapy compared with patient with EFGR

gene mutation. By injecting chemotherapeutic drugs

to patients with different gene mutations, it is found

that the response rate is highest in patients with EGFR

and lowest in patients with EML4-ALK fusion gene.

2.4 Single Agent Chemotherapy and

Combination Chemotherapy

Maio et al. conducted a meta-analysis aimed to

compare the effectiveness of single agent

chemotherapy and combination chemotherapy as a

second-line treatment (Di Maio et al. 2009). The

result shows that as a second-line treatment, the

response rate of patients toward combination

chemotherapy is significantly higher than who

received single agent chemotherapy. However, the

overall surviving rate was not improved. In addition,

most of the patients will end up by developing

resistance to those drugs. Therefore, an innovative

and effective way of treating lung cancer is in urgent

needs.

3 TRADITIONAL TREATMENTS

Due to lung cancer’s high prevalence rate among the

world, a variety of innovative and effective therapies

are in urgent need.

3.1 Chemotherapy

Chemotherapy is the initial treatment that SCLC

patient usually received. It is effective in relieving

various lung cancer complications like bronchial

obstruction, pleural effusion, tumor metastasis and so

on. It is a preferable palliative treatment. Platinum

drugs are the most commonly used chemotherapeutic

drugs on treating lung cancer. It works by bind with

the N7 atom on adenine and guanine to stop DNA

replication and in this way, induce the process of

apoptosis (Rossi & Di 2016). There’re several types

of chemotherapy.

4 ADOPT CRISPR SYSTEM IN

THE TREATMENT OF LUNG

CANCER

CRISPR Cas9 technology is a revolution technique

that allows scientists to target and edit a specific

sequence of genes precisely (

Jiang & Doudna 2017).

Theoretically, it’s possible to delete any cancerous

genes by adopting CRISPR technology. This fresh

idea of treating lung cancer by using “genetic

scissors” has become to a hot topic to conduct

research about recently. In the following section,

different mechanisms of using CRISPR technology to

edit cancer genes will be discussed.

4.1 Delete Cancer Gene

Crispr system takes advantages from those

characteristics of cancer to fight against lung cancer.

By deleting oncogene, activating tumor suppresser

gene, or enhancing the sensitivity of

chemotherapeutic drugs, the syndrome of lung cancer

can be greatly relieved.

In order to use CRISPR Cas9 system to target this

mutated gene, it’s necessary to make an assumption

that there’s a protospacer- adjacent motif (PAM)

sequence which is a nucleotide sequence targeted by

Cas9 nuclease beside the missense gene. By

Adopting CRISPR-mediated Genomic Editing Technique on the Treatment of Lung Cancer: Using Revolutionary Genomic Editing

Technique to Treat Serious Human Disease

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transferring an oncogenic mutant specific Cas9 using

adenovirus as a media, the mutated gene can be

targeted and deleted with high accuracy, which leads

to a significant reduction of tumor production.

4.1.1 Repairing EGFR Gene

In order to treat patients by repairing EGFR gene, the

biopsy sample of patient’s tumor will be first

obtained. The mutated EGFR gene will be identified

from the biopsy and the correspondent single guide

RNA (sgRNA) which is the RNA that will guide the

endonuclease will be designed. The designed sgRNA

will target a

specific region on the mutated exon, e.g.,

L858R in exon 21, E19del in exon 19 and so on.

Then, the sgRNA will guide Cas9 nickase to the

target region which will make a single strand breaks

in on each opposite side of the mutated exon (Fig 4).

Next, the donated healthy DNA sequence will be

substituted to the removed sequence by homology-

directed repair (HDR) where its left and right arms

are connected with the cut, mutated DNA sequence.

The deletion of mutated exon and replacement with

healthy DNA sequence will deracinate the mutated

sequence and thus, stop the progression of lung

cancer. While it is also possible to conduct a non-

homologous end-joining (NHEJ) in which the

mutated sequence is deleted, and rest of the DNA

sequences are rejoined. By those two means, a stop

codon introduced by HDR or indel made by NHEJ

will destroy the translation of EGFR protein. As the

result, the translated protein will lose its normal

function, and therefore will not cause any oncogenic

symptoms (Tang & Shrager 2016).

Figure 4: CRISPR Cas9 nickase cutting the opposite side of

the mutated sequence.

4.1.2 Rearranging EML4-ALK Fusion Gene

Maddalo et al. (2014) conducted an experiment on

adult mice aimed to investigate the efficiency of

CRISPR Cas9 system on rearranging EML4–ALK

oncogene. The study is unusual since it did the

verification in a regressive way rather than a

progression way. This means that instead of

removing the EML4–ALK oncogene in mouses with

lung cancer, they chose to introduce lung tumor into

mouses body. If researcher can indeed add EML4-

ALK fusion gene into mouses genome, it implies that

it’s also possible to remove the oncogene away from

their genome.

They’ve genetically engineered mouses genome

to simulate the most common type of EML4–ALK

variant in NSCLC cases. They did this by introducing

a double stranded DNA that breaks at specific regions

in this case, intron 14 of EML4 gene and intron 19 of

ALK oncogene. Next, in order to express CAS9 and

sgRNA, researcher genetically engineered the

plasmid started from tandem U6 promoters. Then,

they made a recombinant adenovirus (Ad-EA) by

introducing the CAS9 nuclease and sgRNA into an

adenoviral shuttle vector to target the EML4 and

ALK loci. Numerous mouses were infected by Ad-

EA which leads to a speedy production of EML4-

ALK inversion. This is exactly the pathological

causes of NSCLC. After a month of infection, mouses

lungs started to appear several small lesions. By the

time of 6-8 weeks infection, the lungs tumor was

large enough to be easily seen by necropsy and micro-

computed tomography (Fig 5). The result shows that

there’s an obvious appearance and enlargement of

lungs tumor in mouses lungs. This indicated the

succession of rearranging EML4-ALK

fusion gene. It

also implies the feasibility of treating lung cancer by

rearranging EML4-ALK oncogene in a positive way.

Figure 5: The above shows the micro-computed

tomography of mouses lung tumor after 6-8 weeks of Ad-

EA infection, while the below image shows the necropsy of

mouses lung tumor. Ad-Cre is another type of infection that

has the similar mechanism with Ad-EA infection.

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5 CONCLUSIONS

Overall, CRISPR technology is a booming and

innovative gene editing technology. It has an

excellent applicational potential on treating and

investigating lung cancer. CRISPR system can help

scientists to construct tumor model, to study the

pathology of lung cancer, to discover certain drug

resistance and so on. It also enables researcher to

target specific oncogene or tumor suppressor gene.

By deleting or reactivating those genes, cancer

symptoms can be greatly relieved. Recently, there’re

a huge amount of research of adopting CRISPR

system on treating mouses with lung cancer. In order

to apply this system on curing genuine human cases,

more studies are in urgent needs. CRISPR system

must pass several strict assessments before they’re

applied into clinical trials. In addition, there’re a lot

of ethical issues raised in testing the efficiency of

CRISPR system on human samples. More animal

studies are required before it is used in clinical

circumstances.

However, it is necessary to aware the potential

defects of CRISPR technology. Since human genome

is an extremely large system, it is possible for

CRISPR system to cut other genes that are not

targeted by the nucleases which might cause an

unpredictable effect.

This off-targeted effect can be reduced by

improving guide RNA. Studies show that the length

of guide RNA may induce certain type of mutations.

Appropriate length of guide RNA is necessary for an

optimum genome-editing efficiency. Research also

reveals that specific chemical modification of guide

RNA, like introducing 2ʹ-O-methyl-3ʹ-

phosphonoacetate in the sugar backbone of guide

RNA, can significantly reduce the rate of off target

cleavage.

It is also possible to reduce off-target effect by

improving the delivery system of CRISPR

technology. For instance, adeno viruses have the

potential of integrating into target cell genome in a

meager way, which will restrict off-target

influence. In addition, deliver CAS9 protein and

guide RNA together as a ribonucleoprotein complex

will reduce the probability of missing the target gene,

too. Yet, those methods are only be proven by a few

studies. In order to further decrease the rate of off-

target effect, more empirical evidence is needed.

Until now, substantial amount of research has

done on animal sample. Once the technique has

proven to be safe enough, clinical research should be

conducted on human sample in order to adopt

CRISPR technology on treating genuine human lung

cancer. However, researchers should always be aware

of the ethics involved in clinical research and should

strictly follow the ethical guidelines.

REFERENCES

Di Maio, M., Chiodini, P., Georgoulias, V., Hatzidaki, D.,

Takeda, K., Wachters, F. M., ... & Gridelli, C. (2009).

Meta-analysis of single-agent chemotherapy compared

with combination chemotherapy as second-line

treatment of advanced non-small-cell lung cancer. J

Clin Oncol, 27(11), 1836-1843.

Jiang, C., Lin, X., & Zhao, Z. (2019). Applications of

CRISPR/Cas9 technology in the treatment of lung

cancer. Trends in molecular medicine, 25(11), 1039-

1049.

Jiang, F., & Doudna, J. A. (2017). CRISPR–Cas9 structures

and mechanisms. Annual review of biophysics, 46,

505-529.

Koivunen, J. P., Mermel, C., Zejnullahu, K., Murphy, C.,

Lifshits, E., Holmes, A. J., ... & Jänne, P. A. (2008).

EML4-ALK fusion gene and efficacy of an ALK kinase

inhibitor in lung cancer. Clinical cancer research,

14(13), 4275-4283.

Koo, T., Yoon, A. R., Cho, H. Y., Bae, S., Yun, C. O., &

Kim, J. S. (2017). Selective disruption of an oncogenic

mutant allele by CRISPR/Cas9 induces efficient tumor

regression. Nucleic acids research, 45(13), 7897-7908.

Kuramochi, M., Fukuhara, H., Nobukuni, T., Kanbe, T.,

Maruyama, T., Ghosh, H. P., ... & Murakami, Y.

(2001). TSLC1 is a tumor-suppressor gene in human

non-small-cell lung cancer. Nature genetics, 27(4),

427-430.

Maddalo, D., Manchado, E., Concepcion, C. P., Bonetti, C.,

Vidigal, J. A., Han, Y. C., ... & Ventura, A. (2014). In

vivo engineering of oncogenic chromosomal

rearrangements with the CRISPR/Cas9 system. Nature,

516(7531), 423-427.

Morodomi, Y., Takenoyama, M., Inamasu, E., Toyozawa,

R., Kojo, M., Toyokawa, G., ... & Ichinose, Y. (2014).

Non-small cell lung cancer patients with EML4-ALK

fusion gene are insensitive to cytotoxic chemotherapy.

Anticancer research, 34(7), 3825-3830.

Ng, S. R., Rideout, W. M., Akama-Garren, E. H., Bhutkar,

A., Mercer, K. L., Schenkel, J. M., ... & Jacks, T.

(2020). CRISPR-mediated modeling and functional

validation of candidate tumor suppressor genes in small

cell lung cancer. Proceedings of the National Academy

of Sciences, 117(1), 513-521.

Niederhuber JE, et al., eds. Cancer of the lung: Non-small

cell lung cancer and small cell lung cancer. In:

Abeloff's Clinical Oncology. 6th ed. Elsevier; 2020.

https://www.clinicalkey.com. Accessed Jan. 13, 2020.

Non-small cell lung cancer. National Comprehensive

Cancer Network.

https://www.nccn.org/professionals/physician_gls/def

ault.aspx. Accessed Jan. 13, 2020.

Adopting CRISPR-mediated Genomic Editing Technique on the Treatment of Lung Cancer: Using Revolutionary Genomic Editing

Technique to Treat Serious Human Disease

173

Rossi, A., & Di Maio, M. (2016). Platinum-based

chemotherapy in advanced non-small-cell lung cancer:

optimal number of treatment cycles. Expert review of

anticancer therapy, 16(6), 653-660.

Soda, M., Choi, Y. L., Enomoto, M., Takada, S.,

Yamashita, Y., Ishikawa, S., ... & Mano, H. (2007).

Identification of the transforming EML4–ALK fusion

gene in non-small-cell lung cancer. Nature, 448(7153),

561-566.

Tang, H., & Shrager, J. B. (2016). CRISPR/Cas‐mediated

genome editing to treat EGFR‐mutant lung cancer: a

personalized molecular surgical therapy. EMBO

molecular medicine, 8(2), 83-85.

CAIH 2021 - Conference on Artiﬁcial Intelligence and Healthcare

174