CRISPR/Cas9‑Driven Precision Oncology for Lung Cancer: Current

Situation and Challenges

Wenyu He

College of Chemical Engineering, Nanjing Forestry University, 210037, China

Keywords: CRISPR/Cas9, Lung Cancer, Precision Oncology.

Abstract: Different types of lung cancer are treated in different ways. How to choose accurate treatment plan and reverse

drug resistance after treatment will become the key to further improve the prognosis of lung cancer in time to

come. CRISPR/Cas9 gene editing instrument. It capitalizes on the natural immune defense system of

prokaryotes to accurately modify the target gene, compared to other, the design is simpler and lower cost,

only the synthesis of a specific gRNA can target any gene sequence, a wider range of applications. This

technology has displayed the great promise in the field of lung cancer study and the treatment, but it still needs

to face bottlenecks such as off-target effects and delivery systems, and its actual clinical effectiveness needs

to be rigorously verified by more in-depth studies and large-scale clinical trials.

1 INTRODUCTION

Lung cancer ranks as the most common and lethal

malignancy globally, accounting for the highest

burden and mortality worldwide, which has emerged

as a global health crisis, topping the charts as the most

common cancer type and casting a long shadow over

human health.

Although the current diagnosis and treatment

technology has been improved, there is still a lack of

early clinical diagnosis of lung cancer. Most patients

suffering from this malignant disease have no obvious

clinical symptoms, and once diagnosed, most of the

patients have poor survival prognosis due to late

staging, missing the best time for treatment, resulting

in no significant improvement in the 5-year survival

rate.

Conventional therapies to treat lung cancer

include surgical excision, chemotherapy, and

radiotherapy etc. Surgical excision, curative for early-

stage disease, is often infeasible for advanced lung

cancer patients due to the low early diagnosis rate

(only 15%) and advanced-stage presentation in 75%

of patients at diagnosis (Memi F,2018). Even if the

patient meets the surgical indications, irreversible

lung function impairment or inevitable postoperative

complications may result.

Chemotherapy, which serves as the cornerstone of

treatment for advanced - stage patients who are

ineligible for surgical resection, frequently runs into

the roadblock of drug resistance, which not only

undermines the effectiveness of chemotherapy but

also leads to subpar treatment outcomes (Ren

F,2024).

For Radiotherapy, despite its role in reducing

thoracic recurrence. Its survival benefit is modest (5%

improvement at three years) and age-dependent,

showing no efficacy in patients over 70 and potential

harm in older subgroups. Trial heterogeneity—

including variations in radiation doses, timing, and

combination strategies—further complicates its

clinical utility. It does not respond to distant

metastases and may promote the spread of tumor cells

due to the "distal effect". Additionally, radiotherapy’s

nonlethal toxicity profiles remain incompletely

characterized, and long-term follow-up data beyond

three years are limited, leaving uncertainty about its

durability. All of these challenges underscore the

need for innovative approaches to enhance treatment

precision and overcome the resistance mechanisms.

The gene - editing approach founded on CRISPR

and Cas systems has ushered in a novel era for lung

cancer research. The first-in-human Phase I trial

results demonstrated that CRISPR/Cas9-edited PD-1-

targeted T cell therapy in lung cancer patients is

generally safe and feasible. This landmark study

establishes a critical translational foundation for

advancing CRISPR-based gene editing technologies

into subsequent clinical investigations (Lu Y,2020).

368

He, W.

CRISPR/Cas9-Driven Precision Oncology for Lung Cancer: Current Situation and Challenges.

DOI: 10.5220/0014493500004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 368-373

ISBN: 978-989-758-789-4

Distinguished from earlier gene-editing tools like

zinc finger nucleases (ZFNs) and transcription

activator-like effector nucleases (TALENs), the

CRISPR system has established itself as the

preeminent gene-editing platform with the highest

clinical translatability. This technological advantage

stems from its dual attributes: simplified operational

process and unprecedented targeting precision. By

enabling site-specific genomic modifications with

high efficiency, CRISPR technology has not only

revolutionized oncogenesis research but also

unlocked innovative strategies for personalized

cancer treatment. Its capacity to precisely manipulate

disease-causing genes positions it as a cornerstone

technology in the advancement of precision medicine

(Wang SW,2022; Li J,2013).

This paper will explore CRISPR/Cas9’s role in

lung cancer therapy by examining its technical

principles, preclinical breakthroughs in oncogene

editing and drug resistance reversal, and translational

challenges including delivery efficiency, off-target

risks, and ethical considerations, to assess its potential

as a transformative therapeutic approach.

2 TECHNICAL BASIS OF

CRISPR/Cas9

CRISPR is a sequence consisting of a number of

repeated sequence regions and intervals arranged

alternately. Where, the sequence of the repeating

sequence area is arranged in palindromic manner. The

sequence of the spacer region is random, it is derived

from the foreign viral DNA sequence and used to

recognize this sequence when the foreign virus re-

invades, and the Cas protein will interrupt the viral

sequence at this time, thereby providing acquired

immunity to bacteria and archaea. CRISPR/Cas9 uses

a specific RNA to guide endonucrenase to a target,

enabling DNA editing (Nussenzweig PM,2020).

It is taxonomically categorized into Class 1 and

Class 2, with the latter distinguished by simpler

functional protein architecture compared to multi-

protein complexes in Class 1 systems. Among Class

2, Type II Cas9 and Type V Cpf1 represent single-

component nuclease effectors (Memi F,2018). In

general, the core of the class 1 is a complex composed

of proteins such as the helicases Cas3 and Cas8 and

the polymerase Cas10. The second key type of

CRISPR-Cas is distinguished by its unique functional

proteins, namely single CrRNA-binding proteins with

multiple domains. This binding protein internally

integrates all the components needed to perform

nucleic acid cleavage. It can do with only one group

the work that the first class of systems requires

multiple groups to do together

Adaptive immunity mediated by CRISPR/Cas

systems progresses through three conserved phases:

1) foreign DNA derived from invading phages or

plasmids is captured and integrated as spacer arrays

flanked by palindromic repeats into host CRISPR

loci, establishing sequence-specific immunity; 2)

transcription of CRISPR loci generates pre-crRNA,

which undergoes maturation via RNase III cleavage

directed by trans-activating tracrRNA transcribed

from upstream regions, forming a functional

tracrRNA: crRNA duplex; 3) this ribonucleoprotein

complex guides Cas9 nuclease to introduce double-

strand breaks (DSB) at complementary protospacer

adjacent motif (PAM)-containing genomic sites,

synergistically disrupting invading nucleic acids.

Compared to other gene editing technologies, this

system has the highest targeting efficiency (50% to

80%) through preferential binding to open chromatin

regions and utilization of truncated sgRNA (2-3nt

shortening). The core advantage of CRISPR/Cas9

over other genome editing tools lies in its unique

RNA-guided targeting mechanism: target DNA

recognition is achieved via 20-nucleotide sgRNA

base pairing, avoiding the technical bottlenecks of

ZFNs and TALENs that rely on complex DNA-

binding protein design, significantly reducing design

complexity and implementation costs. This system

supports highly efficient multiplexed genome editing,

enabling

simultaneous multi-site editing in

mammalian cells and other systems through co-

delivery of multiple gRNAs, with efficiency far

surpassing traditional tools. Additionally,

CRISPR/Cas9 achieves 1500-fold improved DNA

specificity compared to wild-type Cas9 through

optimized PAM sequences (e.g., CGGH consensus)

combined with Cas9 nickase strategies (e.g., D10A

mutants). Targeting efficiency is further enhanced

permanent gene modification at the DNA level

independent of cell cycle phase; and enhanced

precision with off-target effects reduced to <0.1%

through optimized sgRNA design and homology-

directed repair (HDR) integration (Karimian

A,2019). These technical advancements have

propelled CRISPR-Cas9 to the forefront of

translational applications, particularly in precision

oncology for diseases such as lung cancer, where its

modularity and efficiency enable targeted therapeutic

interventions (Memi F,2018).

CRISPR/Cas9-Driven Precision Oncology for Lung Cancer: Current Situation and Challenges

369

3 CURRENT PROGRESS

3.1 Genome Ablation

In a study, researchers exploited the mutation-

generated protospacer-adjacent motif (PAM, 5’-

CGG-3’) of the oncogenic EGFR L858R mutation

(CTG→CGG) in NSCLC. The researchers designed

a specific sgRNA and delivered CRISPR/Cas9 via

adenoviral vectors (Ad/sgEGFR + Ad/Cas9). This

landmark research, which was published in Nucleic

Acids Research in 2017, first established the in - vivo

proof - of - concept for allele - specific CRISPR/Cas9

- mediated oncogene ablation. Findings indicated that

CRISPR/Cas9-mediated ablation of the L858R

mutation in EGFR-upregulated lung cancer cells

suppressed tumor cell proliferation. This study

addressed precision oncology challenges and

provided a template for personalized therapies

targeting PAM - generating mutations (Karimian

A,2019).

Findings from a human Phase I trial

(NCT02793856) evaluated CRISPR-Cas9-

engineered T cells with PD-1 gene modulation in

individuals with advanced NSCLC. Researchers

introduced plasmids carrying Cas9 and sgRNA into

patients’ T cells. In order to achieve accurate

knockout of the PD-1 gene, analysis of results

revealed that the condition of 8 patients showed a

notable stable trend (Lu Y,2020).

3.2 Genome Cleavage

A study advanced allele-specific CRISPR/Cas9 for

Non-Small Cell Lung Cancer (NSCLC) by targeting

Epidermal Growth Factor Receptor (EGFR) L858R

mutations (T→G transversion). The strategy

exploited the mutation-generated PAM (5’-CGG-3’)

to design a specific sgRNA, delivered via lentiviral

vectors. In vitro, this system achieved 37.9% target

specificity in mutant cells (NCI-H1975), confirmed

by digital PCR and T7 endonuclease assay.

Functional studies showed selective EGFR

downregulation, reduced proliferation (MTT/colony

formation, p<0.0005), and smaller tumor growth in

vivo (p=0.015). The approach targets between 15%

and 35% of cancer mutations characterized by C>G,

A>G, and T>G nucleotide substitutions. And

highlights PAM-dependent specificity as a key

precision oncology tool (Koo T,2017).

3.3 Reduction of Drug Resistance

Gefitinib is used as a linear epidermal growth

receptor - tyrosinase suppressant (EGFR-TKIs) in the

treatment of lung cancer patients after a period of

acquired resistance. The main mechanism is the

secondary mutation of T790M at the second site of

EGFR, which increases the affinity between ATP and

EGFR-TKIS junction domain, resulting in EGFR-

TkIs cannot effectively block the signal pathway and

produce drug resistance.

Guernet and colleagues combined single guide

RNA with single-stranded DNA fragments carrying

unique genetic tags, can precisely track thousands of

tumor clones at the single-cell resolution. It has

successfully constructed resistance models driven by

EGFR T790M/KRAS G12D mutations and those

related to EML4-ALK rearrangements, confirming

that drug resistance can be dynamically formed

through multiple mechanisms such as gene

amplification and bypass signaling activation, which

have made significant breakthroughs in NSCLC drug

resistance research. By simulating the clonal

interactions in the tumor microenvironment, this

system provides innovative tools for optimizing

personalized treatment strategies, effectively

evaluating combination therapies, and deciphering

the special drug resistance mechanisms in patients

with EGFR/ALK co-mutations (Cheung AH,2018;

Marino FZ,2017).

3.4 Economic Efficiency

The continuous innovation of CRISPR-Cas9

technology has brought new hope for alleviating the

economic burden on lung cancer patients. A team led

by Irene Lara-Sáez developed a cationic polymer

delivery system for CRISPR/Cas9 ribonucleoprotein

(RNP), enabling simpler and cost-effective

construction of lung mutation models using chemical

vectors and off-the-shelf synthetic reagents. This

virus-free approach significantly reduces the

technical barriers and production costs of gene

editing. Its precision in inducing genetic

modifications not only allows efficient screening of

oncogene combinations but also expands therapeutic

possibilities beyond traditional gene inactivation. As

this technology becomes widely adopted in cancer

modeling, it will drive rapid iterations of personalized

treatment protocols, ultimately mitigating long-term

economic burdens on lung cancer patients through

dual pathways of improved treatment efficiency and

shortened R&D cycles (Guernet A,2016).

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4 CURRENT LIMITATIONS

4.1 Off Target Effects

The mismatch between the designed specific sgRNA

and the non-target DNA sequence leads to

unexpected mutations in the gene locus, which is

called off-target effect. Off-target will not only

reduce the efficiency of genome editing, but also lead

to the rearrangement of chromosomes, and even

destroy genes that are not perfectly matched, and may

also lead to the inactivity of functional genes.

Within CRISPR/Cas9-based genome editing for

lung cancer, the origination of off-target genomic

alterations is governed by multi-dimensional

regulatory networks. The mismatching between

sgRNA and target sequence is the key factor that

causes the off-target phenomenon.The effective

concentration of Cas9/sgRNA complexes exhibits a

concentration-dependent relationship with cleavage

specificity - when complex concentrations exceed

physiological thresholds, Cas9 cleavage specificity

decreases significantly. This concentration-

dependent risk is particularly prominent in lung

cancer therapy, as high-dose delivery strategies are

often employed to overcome the low transfection

efficiency of tumor cells. PAM sequence, length and

adjacent seed region can affect the missed target to

some extent. In addition, the structure, shear activity

and plasmid transfection concentration of Cas9

affected the off-target to some extent. Notably, the

type of transfected cell is also a key factor affecting

off-target.

From the sgRNA point,it was confirmed that the

accuracy and specificity of sgRNA gene editing were

proportional to GC content in sgRNA seed region,

and when GC content was 40% ~ 60%, off-target was

not easy to occur (Ren X,2014). When two G bases

are added to the 5 'terminal of the sgRNA, the

incidence of off-target events can be reduced. In

addition, the commonly used sgRNA guidance

sequence composed of 20 nucleotides was shortened

by 2 to 3 nucleotides, which could not only ensure the

efficiency of targeting binding sites, while also

reduce the occurrence of off-target. From the cas9

point, the fusion protein d Cas9-Fok I monomer has

the cutting ability only when it is combined with two

Sgrnas, thus forming a correct double-stranded

incision and reducing off-target (Lara-Sáez,2024;

Pattanayak V,2013).

4.2 Delivery Challenges

At present, in vitro delivery technology has been

developed to some extent, while there are still some

limitations, likephysical methods (e.g.,

electroporation, liposome transfection) are simple

and efficient in vitro, but hydrodynamic injection

causes liver damage and cardiovascular dysfunction;

viral vectors (e.g., adenoviral, AAV, lentiviral) are

among the earliest developed delivery technologies,

emerging in the 1990s. Their delivery efficiency is

high, but there are potential risks of immunogenicity,

insertion mutations, and high off-target effects, which

limit their further application. Nonviral vectors (e.g.,

lipid-based/polymer-based nanocarriers) may

effectively solve the problems of potential toxicity

and capacity limitation caused by viral vectors. And

has the advantages of simple and easy to obtain, low

cost, safety and so on. Among them, the strategy

based on liposomal delivery of Cas9 mRNA/sgRNA

has entered the clinical experimental stage,further

propels the implementation of CRISPR/Cas9 genome

editing vectors in creating innovative gene therapy

modalities.The novel biocompatible, non-

immunogenic and biodegradable non-viral

nanomaterials are ideal vectors to reduce the risk of

organ toxicity and local inflammation. It will provide

a new impetus for the targeted delivery and clinical

conversion of CRISPR/Cas9 systems in the future

(Wu X,2014).

4.3 Ethical Concerns

CRISPR/Cas9 raises complex ethical considerations,

with core controversies centered on technical risks,

clinical trial design, stakeholder influences, and

patient autonomy.

The technology’s potential risks in somatic gene

therapy—including off-target effects, mosaicism

formation, and uncertainties regarding long-term

safety—stand in stark contrast to the reversibility of

traditional drug trials. For example, it may induce

new cancers or genetic defects.And its irreversibility

and "all-or-nothing" nature pose unique challenges

for clinical trial design, necessitating phased

monitoring mechanisms and reliance on extrapolation

of primate experimental data, yet existing regulatory

frameworks remain ill-adapted to such novel

therapies (Li L,2015).

Debates also revolve around the ethical boundary

between gene therapy and enhancement. While lung

cancer treatment falls within the scope of disease

correction, the technology’s efficiency may blur the

CRISPR/Cas9-Driven Precision Oncology for Lung Cancer: Current Situation and Challenges

371

line between therapeutic and optimizing

interventions. Historical precedents (e.g., IVF and

mitochondrial transfer) demonstrate shifting public

acceptance with accumulating evidence of medical

benefits, but CRISPR/Cas9’s non-therapeutic

applications in animal models—such as antiviral

resistance and muscle enhancement—hint at potential

"slippery slope" risks (Baumann M,2016).

Stakeholder dynamics are critical: pharmaceutical

companies may emphasize benefits under economic

pressures, while public sensitivity to issues like

"designer babies" could lead to either exaggerated

fears or underestimation of risks in the oncology

context. Additionally, the high costs of genetic

screening and insurer payment restrictions may force

patients to assume treatment risks with limited

information, exacerbating tensions between patient

autonomy and medical decision-making. Therefore, it

is necessary to establish a rigorous ethical review

mechanism for clinical trials to ensure that the risk-

benefit ratio is reasonable and adopt a dynamic

informed consent model to allow patients to continue

to participate in decision-making during treatment.

5 CONCLUSION

CRISPR/Cas9 has established itself as a

revolutionary tool in precision oncology, particularly

in lung cancer treatment, by enabling unprecedented

gene-editing capabilities. Current progress highlights

its utility in oncogene ablation (e.g., EGFR

mutations), overcoming drug resistance through

clonal profiling, and generating genetically

engineered preclinical models. However, technical

bottlenecks remain significant hurdles. Ethical

considerations, such as the risk of therapeutic creep

toward enhancement and regulatory gaps, further

complicate its implementation.

Looking ahead, advancements in CRISPR/Cas9

for lung cancer will likely focus on three key areas.

First, improving targeting precision through

computational sgRNA design tools and high-fidelity

Cas9 variants could mitigate off-target risks. Second,

developing novel delivery systems—such as cell-

penetrating peptides, organ-specific nanocarriers, or

viral vectors with reduced immunogenicity—will be

critical for achieving efficient and safe in vivo

genome editing. Third, integrating CRISPR/Cas9

with emerging therapies (e.g., immunotherapy,

targeted kinase inhibitors) may synergistically

enhance therapeutic outcomes while addressing

tumor heterogeneity.

To address these challenges and opportunities,

broad cooperation and joint efforts from all sectors of

the world have become essential. It is necessary to

strengthen international cooperation to develop more

detailed and comprehensive legal regulations to

ensure the healthy development of this technology

under the double framework of moral and legal law.

At the same time, it is also necessary to ensure the

security and controllability of the technology. In

addition, public participation is also indispensable,

through enhancing social ethical and moral awareness

and scientific literacy, jointly promote the rational

application of gene editing technology.

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