Mechanisms Insights into the Role of CRISPR/CAS9 in Breast

Cancer Development

Ruoyu Zhou

College of Marine Life Sciences, Ocean University of China, Qingdao, China

Keywords: CRISPR/Cas9, Breast Cancer, Gene Editing.

Abstract: The function of CRISPR/Cas9 gene editing technology in the evolution and therapy of breast cancer is

discussed in this paper. Although existing therapy approaches have many flaws and breast cancer is a high-

incidence malignancy in women worldwide, CRISPR/Cas9 technology offers a fresh approach with its

precision editing capacity. This paper presents the fundamental idea, method of delivery, and application of

CRISPR/Cas9 technology in the treatment of breast cancer together with targeted resistance mechanism,

precision therapy, and immune cell modification. Simultaneously, the obstacles that the technology faces—

off-target effect, immune response and delivery efficiency—are covered, and potential development paths are

hinted at. Expected to be a major turning point in breast cancer treatment, CRISPR/Cas9 technology will

provide accurate and successful treatment plans to patients and help to prevent and treat breast cancer to a

new age.

1 INTRODUCTION

In contemporary society, cancer has emerged as a

global health crisis, particularly as the second

leading cause of death in the United States (Siegel

et al. 2017). Among various cancers, breast cancer

stands out as one of the most prevalent types and a

significant contributor to female mortality

(Curigliano 2012, Siegel et al. 2022). In Western

countries, breast cancer is among the most

frequently diagnosed diseases among women, with

the highest incidence rates observed in the

European region (Bodewes et al. 2022). Given these

alarming statistics, enhancing the prevention of

breast cancer and identifying novel therapeutic

approaches are not only inevitable imperatives of

our time but also hold profound scientific

significance.

Although traditional cancer treatments, such as

chemotherapy, cytotoxic therapy, and surgical

resection, are widely used, they are often associated

with significant drawbacks, including severe side

effects and a high risk of complications (Yang et al.

2021). CRISPR/Cas9 is a highly innovative gene-

editing technology that enables the correction,

insertion, or deletion of genetic material in both

vivo and in vitro settings (Karn et al. 2022). This

technology employs the principle of primer

targeting to identify specific DNA sequences and

guides the Cas protein to a precise location in the

genome by modifying a guide RNA sequence,

thereby enhancing the efficiency of gene editing (Li

et al. 2023). In breast cancer research,

CRISPR/Cas9 has been instrumental in elucidating

the mechanisms of drug resistance and immune

evasion in tumor cells. It has emerged as an

extremely important, effective, and direct tool in the

treatment of breast cancer (Sabit et al. 2021).

Breast cancer can be induced by genetic

mutations or familial inheritance, as well as

alterations in the microenvironment of breast cells

(Obeague & Obeague 2024). However, despite

significant advancements in breast cancer research,

its complex pathogenesis remains incompletely

understood. Additionally, breast cancer is highly

heterogeneous, with distinct subtypes exhibiting

marked differences in biological behavior and

treatment response (Wang et al. 2024). Therefore,

systematically elucidating the role of CRISPR/Cas9

technology in the development and progression of

breast cancer, and summarizing relevant research

progress, are of great significance for deepening our

understanding of its pathological mechanisms and

identifying new therapeutic targets and strategies.

Through a comprehensive literature review, the

162

Zhou, R.

Mechanisms Insights into the Role of CRISPR/CAS9 in Breast Cancer Development.

DOI: 10.5220/0014439900004933

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 162-167

ISBN: 978-989-758-789-4

current state and limitations of the existing research

can be better understood, providing guidance for

future research directions and advance the

development of breast cancer prevention and

treatment strategies.

2 FUNDAMENTAL PRINCIPLES

OF CRISPR/CAS9

TECHNOLOGY

As an epochal gene editing tool, CRISPR/Cas9

employs the complementary effects of gRNA and

Cas9 endonuclinase to precisely change a genome.

With 20 nucleotide sequences, gRNA’s sequence is

straightforward, and the 20-base spacer sequence is

supplementary coupled with the target DNA during

the working process so directing Cas9 to a specific

genetic spot. This process reflects the high specificity

of genes (Asmamaw＆Zawdie 2021). After that, the

single-stranded guide RNA, or gRNA, hooks itself to

the Cas9 nuclease to create a CRISpen-Cas9

functional complex. To locate the proto-spacer

sequence adjacent motif (PAM) at the 3’ end of the

target sequence, the complex glides down the DNA

strand (Aljabali et al. 2024). The PAM sequence is

the fundamental component for Cas9 to identify and

bind the target DNA; its presence or absence affects

whether the system can operate as it should. Usually

a short-maintained sequence, the PAM sequence is

"NGG," in the common SpCas9 system, where "N"

indicates any base and "GG" is the complete PAM

sequence. Although only DNA fragments which

includes PAM sequences can be discovered and cut

by Cas9, this conserved PAM sequence offers

unambiguous guidelines for the aiming design of

CRISPR/Cas9 systems and limits their targeting

range. Cas9 starts the DNA cutting process when it

identifies PAM. The conformation of Cas9 changes

when sgRNA complements and pairs with the target

DNA sequence to produce PAM. The alteration of

Cas9 conformation boosts nuclease activity even

further. The Cas9 protein is now creating a double-

strand break (DSB), cutting two strands of the target

DNA throughout the nucleic acid domain. Key to

enabling gene editing using CRISpen/Cas9 is the

formation of double-stranded breaks; thereafter, the

DNA repair machinery within the cell is triggered to

repair DNA via either homologous directed repair

(HDR) or non-homologous end joining (NHEJ).

The non-homologous end-join repair process

(NHEJ) is most notable for without relying on the

homologous template, hence it may be used in most

cell types and occurs during any cycle of cell

division. Although NHEJ maintains DNA rather

quickly, since it does not rely on homologous

templates, the repair results are generally less

precise and can generate either insertion or deletion

modifications. Conversely, homologous directed

repair (HDR) has greater significance in procedures

requiring exact manipulation such as gene insertion,

knockout, and replacement since it utilizes

homologous templates for specific repair. The

technological advances in the complexity of the

HDR technique are higher, nonetheless, and

operating methods and examination circumstances

are more strictly requested. Usually occurring only

in the S and G2 phases of the cell, HDR results from

sister chromatids present within a cell fit for

homologous templates. Moreover, HDR is

somewhat ineffective, particularly in some non-

dividing cells, which restricts its utility in specific

application situations. One can treat breast cancer

using the fundamental ideas of CRISPR/Cas9

technology. Its exact positioning ability assists it to

repair or knock out the genes linked to the

development and progression of breast cancer,

therefore attaining the effect of precision treatment.

Alternatively use its technologies to change

immune cells to improve immune system

performance, thereby preventing the emergence of

breast cancer. The development and creativity of

CRISPR/Cas9 technology offer a wide range of

opportunities for managing the worldwide sickness

of breast cancer.

3 DELIVERY METHODS FOR

CRISPR/Cas9

Once the fundamental ideas of CRISpen/Cas9

technology are grasped, its delivery in the body has

also become a big issue. The most often used methods

of delivery nowadays are plasmids, mRNA,

ribonucleoprotein complexes (RNP), and viruses.

Among multiple forms, the plasmid delivery

technique is the simplest and most reasonably priced;

nonetheless, the transfection efficiency is rather poor

and can cause an immune response or insertion

mutation. Although mRNA delivery can be expressed

quickly and helps to avoid genome integration, its

stability and delivery efficiency still have to be

further improved. Although the flaws of this delivery

technique are that it has poor in vivo stability and

restricted delivery efficiency, ribonucleoprotein

Mechanisms Insights into the Role of CRISPR/CAS9 in Breast Cancer Development

163

complex (RNP) delivery can enable fast gene editing

and lower the chance of off-target effects. Although

viral vectors—such as adenoviruses—have great

transduction capability and adaptability to a broad

spectrum of cell types—there are vector capacity

limits and possible immunological concerns. Thus,

carrying out the use of CRISPR/Cas9 technology in

breast cancer treatment depends mostly on its

intended distribution form. This paper will

extensively address in the sections that follow how to

distribute CRISPR/Cas9 within cells to generate

novel concepts and strategies for the exact treatment

of breast cancer.

3.1 Plasmid DNA Delivery System

By containing the coding sequence of Cas9 nuclease

and gRNA, direct transfection of plasmid DNA is the

easiest approach to introduce external genetic

material and attain long-term stable expression. The

drawback of this method, though, is that Cas9

expression can cause more off-target effects and

insertion mutations. Conversely, random integration

of plasmid DNA can cause insertion mutations, which

would subsequently influence the normal

physiological operation of cells. The researchers will

thus seek to place a degradation signal sequence or

activity inhibition domain on Cas9 in order to lower

these hazards. This ensures exact control of Cas9

protein activity, therefore lowering the possibility of

off-target repercussions and insertion mutations.

Simple and inexpensive to manufacture, plasmids can

be extensively utilized in laboratory research.

Nevertheless, the process of plasmid subsequent

generations and purification has dangers of endotoxin

contamination and host immunological reaction; so,

the presence of endotoxin will set off the host immune

response and influence the outcome of the

experiment. Researchers have lately solved these

issues by creating microcyclic plasmids (Ahmed et al.

2021). Non-viral methods of distribution call for

additional components—physical means

(electroporation, microinjection) or lipid

nanoparticles (LNP)—to aid across the cell

membrane. Under the physical strategy, the

electroporation method uses a brief pulse of high

voltage to generate reversible pores in the cell

membrane, consequently facilitating the entrance of

plasmid DNA. Microinjection is the direct injection

of plasmid DNA into the cytoplasm under a

microscope using microneedles. Though theoretically

the physical technique may significantly boost the

delivery efficiency, it damages cells dramatically and

is usually suitable for single-cell level operation.

Chemical techniques distribute plasmid DNA

primarily through lipid nanoparticles. Including lipid,

lipid nanoparticles are nanometers carriers that can

wrap plasmid DNA and shield it from nuclease

breakdown. By means of fusing with the cell

membrane, lipid nanoparticles can efficiently

introduce plasmid DNA into the cell, hence

improving the efficiency of transport and decreasing

immune response.

3.2 RNA Delivery

Gene editing efficiency and safety render RNA

delivery options based on CRISPR components—

such as Cas9 mRNA in conjunction with gRNA—

better than traditional plasmid DNA delivery. Short-

term studies might consider RNA delivery

appropriate since it uses cellular translation

procedures to rapidly start editing without waiting for

plasmid transcription. Its brief action cycle lowers the

likelihood of genome insertion and off-target

alterations. Additionally, RNA has a brief action

cycle in the cell, which helps significantly decrease

the risk of off-target and undetected risks of genome

insertion mutations, thus ensuring the safety of gene

editing. But the low temperature storage and

transportation demand as well as the great purity

modification of mRNA raise the production cost.

While their mature technology and effective delivery

capability have made lipid nanoparticles (LNP) the

preferred choice as the principal carrier of RNA

shipping, their greater requirements for targeting

accuracy have led to their marginal acceptance.

Studies have revealed, for instance, that package

delivery vectors (EDVs) can greatly lower the

required CRISPR-Cas9 RNP dose and are at least

twice as quick and more efficient than electroporation

delivery CRISPR-Cas9 RNP. Safe and more efficient

delivery technology for gene therapy has resulted

from research on RNA delivery methods. Chemically

modified GRnas can enhance CRISpen-Cas gene

editing efficiency in basic human cells. Furthermore

looked into are the use of biodegradable lipid

nanoparticles for Cas9 mRNA delivery to increase

gene editing efficiency and safety criteria. All things

considered, CRISpen component RNA delivery

methods are somewhat common in the world of gene

editing but also have to balance technical maturity

against safety. More research will maximize the RNA

delivery mechanism to forward the clinical

application of gene therapy.

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3.3 RNP Delivery System

Apart from the above mentioned delivery techniques,

the most effective approach to provide CRISpen/Cas9

components is the ribonucleoprotein complex (RNP).

RNP is a compound pre-assembled by Cas9 protein

and guide RNA (gRNA), so Cas9 protein and gRNA

can straight enter the nucleus and rapidly start gene

editing without the need of transcription and

translation. By entering the nucleus straight and

starting rapid gene editing, the Cas9 protein and

gRNA greatly shorten the start-up time for gene

editing. This approach reduces off-target effects and

minimizes the possibility of foreign genes merging

into the host genome as compared with RNA

delivery. But RNP has higher technical operating

requirements and is less stable than plasmid and

mRNA as protease readily breaks it. Strict oversight

of the concentration, delivery its duration, which stem

and cellular environment of RNP during delivery is

required, for instance, to guarantee its stability and

function in the cell. RNP should thus be investigated

case-by-case since cell type and delivery vector affect

its delivery efficiency and impact it.

4 APPLICATIONS OF

CRISPR/Cas9 TECHNOLOGY

IN BREAST CANCER

THERAPY

Due to its unique and innovative working mechanism,

CRISPR/Cas9 technology has shown great potential

in tumor therapy applications since its invention.

More especially, a discovery has been made about

triple negative breast cancer (TNBC). Triple-negative

breast cancer (TNBC) is one of the most aggressive

tumor subtypes; its high recurrence rate, difficult

prevention, and lack of therapeutic response have

long perplexed people. Therefore, it is essential to

innovate a new type of tumor treatment. Academician

Cao Xuetao’s team discovered the existence of CD28

in cancer cells and its role in tumor therapy and

immunity (Yang et al. 2025).

4.1 Targeting Drug Resistance

Mechanisms

Though in clinical practice some individuals still

display drug resistance at the initial stage or

secondary drug resistance after therapy, at present

with the development of several medications, breast

cancer patients have many alternatives for treatment.

The application of CRISPR/Cas9’s targeted

recognition DNA for gene editing in the treatment of

breast cancer is conducive to the construction of

stable cell lines carrying target gene mutations, and

provides a good platform for screening drug targets.

4.2 Precision Therapy

By identifying and screening gene targets,

CRISPR/Cas9 technology increases the likelihood of

precisely treating TNBC by identifying tumor

inhibitors, oncogenes, and associated drug resistance

genes. In 2016, Chinese scientists used CRISPR/Cas9

to knock out ST8SIA1, a gene that affects TNBC

metastasis and recurrence, in the clinical treatment of

lung cancer, which can effectively inhibit the growth

and spread of its cancer cells. In terms of drug

combination therapy, CRISPR/Cas9 can target

various drug resistance genes, such as knocking out

PARP1 to make cells sensitive to adriamycin,

gemcitabine and other drugs (Vaghari-Tabari et al.

2022). So CRISPR can be combined with drugs to

treat TNBC.

4.3 Future Directions

Many clinical studies are now assessing the safety

and efficacy of CRISpen/Cas9 technology in breast

cancer treatment; a major obstacle is how to prevent

the immune response generated by Cas9 protein in the

clinical process, which can influence the treatment

impact. Studies have shown, for example, that the

Cas9 protein from Staphylococcus aureus and

Streptococcus pyogenes may trigger an immune

reaction in the body, therefore affecting not only the

efficacy of gene editing but also maybe generating

adverse effects. If this issue is to be tackled, future

research should focus on ways to stop immunological

reactions produced by CRISpen/Cas9 technology.

Changing the Cas9 protein or choosing a more

immunocompatible vector could help to lower

immunogenicity. For instance, avoiding humoral and

cellular immunological responses in mice has been

demonstrated by delivery of modified Cas9 proteins

via an AAV (adeno-associated virus) vector.

Moreover, crucial directions for next study are

refining the delivery mechanism to lower untargeted

editing and raise the precision of gene editing.

Simultaneously, the future enhancement of

CRISPR/Cas9 technology requires increasing the

efficiency and safety of gene editing tools. The

growth of high-fidelity Cas enzyme shifts and

Mechanisms Insights into the Role of CRISPR/CAS9 in Breast Cancer Development

165

optimization of editing methods, for instance, can

minimize off-target effects and thus raise editing

accuracy. It also has fresh gene-editing procedures

including Prime Editors and Base Editor.

Consequently, in next research, the main challenge to

be solved is how to prevent the immune response

caused by this technology influencing the therapeutic

effect. The original goal of inventive future

development of CRISPR/Cas9 technology is to

maximize gene editing tools and increase the

efficiency and safety of editing in future studies so as

to deliver fresh cure hopes to breast cancer patients.

5 THE FUTURE OF CRISPR/Cas9

TECHNOLOGY

As a revolutionary technology in the field of life

sciences, CRISPR/Cas9 is poised to demonstrate

broad development prospects and potential across

medicine, agriculture, and basic research. In

medicine, its high-precision gene-editing capabilities

and targeting effects will play a pivotal role in treating

genetic diseases, neurological disorders, cancer, and

more. For instance, in late 2023, the US FDA

approved Casgevy, the first CRISPR-based gene-

editing therapy, for treating sickle cell disease (SCD).

In 2024, it was also approved for transfusion-

dependent beta-thalassemia (TDT) (Parums 2024,

Singh et al. 2024). In agriculture, CRISPR/Cas9 can

enhance crops through gene editing, improving yield,

resistance, and environmental adaptability. However,

the widespread application of this technology will

inevitably raise ethical and moral concerns. As

research progresses, it is essential to strengthen the

ethical guidelines and regulatory frameworks

governing its use. Overall, the future of

CRISPR/Cas9 technology is full of both promise and

uncertainty.

6 CONCLUSION

In addition to discussing possibilities for curing a

number of illnesses, this article examines the

mechanism of action and technological developments

of CRISPR/Cas9 in the occurrence and progression of

breast cancer. In the modern world, breast cancer is a

high-incidence malignancy that presents a threat to

women’s lives and health. When compared to

conventional treatment gets closer, the advent of

CRISPR/Cas9 technology reduced numerous faults,

thanks to its precision treatment features, offering a

novel approach to the treatment of breast cancer. This

work clarifies in great detail the mechanism of

CRISPR/Cas9 technology in vivo. Perfect gene

editing is made possible by the cooperative activity of

Cas9 protein and gRNA. Furthermore to the

mechanism of action, the advantages and limitations

of three CRISPR/Cas9 delivery methods—plasmid

DNA, RNA, and ribonucleoprotein complex

(RNP)—are discussed. Despite being a novel cancer

treatment method, CRISPR/Cas9 faces significant

obstacles due to the high consistency and complicated

biology of breast cancer.

The precise editing of genes by

CRISPR/Cas9 technology may target the knocking

out or repair of genes connected in the development

of tumors, so drastically stopping the spread of

cancer cells in the research of breast cancer

treatment. By means of precision therapy and

targeted resistance mechanisms, this paper shows

that CRISPR/Cas9 technology greatly increases the

sensitivity of drug therapy through triple-negative

breast cancer (TNBC), so offering creative

solutions for subtypes hard to overcome by

traditional therapies. In addition, the method can be

applied to change immune cells to improve their

capacity of recognizing and eliminating cancerous

cells, therefore activating the immunological

defense system.

Though difficulties related to human trials

including off-target effects, easy immune response

and delivery efficiency still exist to CRISPR/Cas9

technology, its enormous future potential in the

treatment of breast cancer has been typically

acknowledged. Future studies ought to focus more

on ways to maximize the technology of gene editing

to raise their efficiency. At the same time, it

enhances the ethical and legal structure to guarantee

the legitimacy and safety of newly developed

technology. With the ongoing development of

technology, CRISPR/Cas9 technology is expected

to be a major breakthrough point in the field of

breast cancer treatment, bringing more accurate,

efficient and personalized treatment plans to

patients, and so promoting breast cancer treatment

to a new era.

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