A Promising Therapy in Cancer Treatment: CRISPR‑Cas9 Gene

Editing, Its Improvements, Combination Therapies, and Future

Developments

Yuman Sui

Qingdao Academy, Qingdao, China

Keywords: CRISPR‑Cas9, Cancer Immunotherapy, CAR‑T Cells.

Abstract: Cancer, which was caused by uncontrollable cell growth, brings pain and despair to patients and their families.

Individuals in the field of cancer biology never stop finding new therapies effective enough to save sufferers

from huge torments. Crispr-Cas9 is characterized by its ability to cut off designated DNA segments.

Consequently, scientists take advantage of it to silence the expression of certain genes suspectable in

triggering the growth of tumor cells. This review is primarily going to focus on explaining what the CRISPR-

Cas9 is, including the origin of CRISPR-Cas9 system and composition of it, and elaborate on its working

mecha-nisms. Then the article will talk about clinical applications, such as CRISPR-Cas9’s role in co-lon

cancer and Gallbladder cancer, and existing challenges from medical, ethical perspectives. Finally, it will

introduce a combined therapy: CRISPR-Cas9 gene editing technology and CAR-T cell immunotherapy in the

treatment of cancer and future development of gene therapy.

1 INTRODUCTION

On February 2, 2024, the World Health

Organization's International Agency for Research

on Cancer (IARC) recently released the news

“Global cancer burden growing, amidst mounting

need for services”, which once again emphasizes

the increasing global cancer burden that deserves

worldwide attention. It points out that in 2022, there

were 20 million new cancer cases and 9.7 million

deaths worldwide. Successful treatment and the

elimination of cancer have been one of the greatest

dreams for doctors and researchers in the medical

field. Scientists conduct tons of investigations on

the invention of new cancer treatments and several

cancer treatments have been discovered. But there

are still issues and deficiencies present in the

traditional therapies, including nonspecific killing

in chemotherapy/radiotherapy which causes normal

cell damage, bottlenecks for the drug resistance in

targeted therapies, such as EGFR-TKI acquired

resistance and side effects observed in ICI therapy.

All of these limitations drive the exploration of

precise in the field of cancer immunotherapy,

further stimulating the research of gene editing

technology. Gene editing, also called genetic

modification, is a set of technologies utilized to

modify the genetic makeup of cells (Hu et al. 2023).

For CRISPR-CAS9(clustered regularly interspaced

short palindromic repeats/CRISPR-associated

protein 9), it is originally found in eubacteria and

arachnoid membranes for resisting the invasion of

bacteriophage, consisted of Cas9 nuclease and

gRNA.

CRISPR loci are transcribed into pre-CRISPR

RNAs(pre-crRNAs) (Meng et al. 2023). These pre-

crRNAs are processed into mature crRNAs through

the action of trans-activating crRNA(tracrRNA),

endonuclease Cas9, and RNase Ⅲ. The mature

crRNA contains a spacer sequence that can

recognize a specific target DNA sequence.

CRISPR-Cas9 system can be referred as a

molecular scissor when mature crRNA pairs with

tracrRNA to form a double-stranded RNA structure

(Feng et al. 2024, Rabaan et al. 2023). There are

clinical applications of CRISPR-Cas9 gene editing

technology. In colorectal cancer therapy, WHSC1

knockout in colon cancer cells inhibits proliferation

and increases drug sensitivity. CYSLTR1 knockout

weakens 5 - FU resistance (Meng et al. 2023, Hu et

al. 2023). Over time, expansions of CRISPR-Cas9

system’s capabilities and combinations of gene

248

Sui, Y.

A Promising Therapy in Cancer Treatment: CRISPR-Cas9 Gene Editing, Its Improvements, Combination Therapies, and Future Developments.

DOI: 10.5220/0014486000004933

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 248-253

ISBN: 978-989-758-789-4

editing technologies with other cancer

immunotherapies such as drugs or virus vaccines

allows for enlargement in the range of applications

(Akhoundi et al. 2021, Razeghian et al. 2021).

However, CRSPR-Cas9 also has deficiencies, such

as specificity and off-target effects, delivery system

limitations and immune response, but more

solutions and improvements will be made by

scientists in the future (Rabaan et al. 2023). This

article describes the basic principles of CRISPR-

Cas9 gene editing technology, how it works as a

treatment of cancers and the applications at clinical

level (Feng et al. 2024). It will also elaborate on

current issues and obstacles of it and some thoughts

on solving the problems. Lastly, the article is going

to show combined therapy for CRISSPR-Cas9 and

future direction in which gene editing technology is

progressing, as outlined below (Liu et al. 2023,

Alaa 2024).

2 THE CRISPR-CAS9 SYSTEM

AND ITS UNDERLYING

PRINCIPLES

Crispr-Cas 9, the Clustered Regularly Interspaced

Short Palindromic Repeat/ CRISPR associated

protein 9, is observed in the genome of Escherichia

coli by Japanese scientists in 1987 at the first time.

The origin of CRISPR-Cas9 can be traced back to the

eubacteria and arachnoid membranes that use this

natural mechanism to disrupt the DNA/RNA of

invading bacteriophage (Meng et al. 2023). There are

three steps involved in the CRISPR-Cas-mediated

adaptive immunity. When the bacterium is infected,

it would “cut” small pieces of DNA form the virus

and insert them into their own DNA. This piece of

genetic material of bacteriophage, named CRISPR

array, enables the bacterium to memorize the virus

(Hu et al. 2023). Second, a bacterium will

immediately recognize the same virus which invades

at the next time by generating RNA segments that can

identify viruses and attach to the DNA region of

viruses. Then the bacterium will disable the virus

using Cas9 or an enzyme to scissor the associated

DNA in the virus (Meng et al. 2023, Hu et al. 2023).

The CRISPR-Cas9 system is categorized into Class I

and Class II. Class I is characterized by the presence

Cas-protein complexes to identify complementary

DNA, whereas the CRISPR-Cas9 system of Class II

does not have to produce the complicated protein

complex (Meng et al. 2023). Considering the

simplicity and convenience, the CRISPR-Cas9

system has been developed and applicated widely in

gene knockout research of different species. The

CRISPR-Cas9 system is composed of CRISPR-

associated proteins (cas-9) and guide RNA (gRNA).

Cas 9 protein, the genetic scissor, is an endonuclease

responsible for double stranded DNA breaking (Feng

et al. 2024). It has the ability to recognize and bind to

the Protospacer adjacent motif (PAM) in the genome,

causing the DNA unwinding. For the gRNA, it

consists CRISPR RNA/crRNA and trans-activating

CRISPR RNA/tracrRNA. crRNA, with the length of

18-20 base pair, is specific to target DNA through

pairing with targeted DNA sequence. TracrRNA is

featured by a long stretch of loops that facilitate the

building for Cas-9 nuclease (Hu et al. 2023).

The mechanisms of CRISPR-Cas 9 gene editing

system are three phases: recognition, cleavage, and

repair. First, the CRISPR-Cas9 system recognizes

PAMs in the DNA of invading virus or

bacteriophage (Meng et al. 2023). The foreign DNA

is cleaved into short spacer sequences, which are

then integrated into the host’s CRISPR array via

Cas1/Cas2 protein complex. These spacers are

inserted between repeat sequences in the array,

forming a genetic memory of the infection, so a

recognition is gained. Second, Cas 9 protein scans

the DNA for the PAM sequence downstream of the

target site. Upon PAM recognition, Cas 9 induces

local DNA unwinding, separating the double helix.

The crRNA region of the sgRNA forms an RNA-

DNA hybrid with the complementary DNA strand

(Meng et al. 2023, Hu et al. 2023). Positioned near

the RNA-DNA hybrid, the HNH domain cleaves the

DNA strand complementary the crRNA. This cut

occurs 3 bp upstream of the PAM. The non-

complementary DNA strand is cleaved by RuvC-

like domain. HNH and RuvC (protein structure

domains of Cas 9) create a blunt-ended double-

strand break (DSB). DSB triggers cellular repair

pathways. There are two pathways: non-

homologous end joining (NHEJ) and homology-

directed repair (HDR). NHEJ gives rise to

deletion/insertion of DNA and keeps active

throughout the cell cycle, while HDR having a

higher genome editing accuracy adds homologous

donor DNA template at the DSB site (Meng et al.

2023).

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3 CLINICAL APPLICATIONS OF

CRISPR-CAS9

Since the scientists have uncovered the basic working

mechanisms of CRISPR-Cas 9 system, they begin

using it as a gene editing technology in the field of

medicine, agriculture and gene modification. This

review will mainly discuss the application of

CRISPR-Cas 9 system in different cancer types.

There are progressions being observed in the use of

gene editing technology to treat colon cancer at

clinical level (Meng et al. 2023, Hu et al. 2023). Gene

knockout, a technique of introducing a mutation in

order to inactivate an organism’s gene that is

suspicious to control a certain trait. Knocking out

PUM1 in cells via CRISPR-Cas 9, scientists find that

the cell activity is reduced by 25-30%. Tumor cell

growth is inhibited when WHSC1 is knocked out and

the metastasis ability is further decreased. The effect

of CRISPR-Cas 9 for gene knockout makes genes

encouraging tumor cells’ growth and providing

proper condition for the development of colon cancer

unable to regulate the expressions anymore (Meng et

al. 2023). Also, the scientists utilize CRISPR-Cas 9

system to construct 3D animal model to stimulate the

tumor environment. Designed sgRNA can induce

particular mutations, enabling the model with specific

genetic mutation to be investigated (Hu et al. 2023).

In the experiment of human intestinal stem cells,

scientists use CRISPR-Cas9 to introduce four

frequently mutated genes (TP53, SMAD4, APC and

KRAS) in activated colorectal tumor cells, observing

the growth of carcinoma.

The strength of CIRSPR-Cas 9 is revealed in

curing gallbladder cancer as well: first, the research

team in Germany utilize Cas9 and other tools to

silence the expression of TP53 and activate latent

KRAS mutant (two most frequently mutated genes

of the Gallbladder cancer) (Erlangga et al. 2019). A

negative control was carried out by an sgRNA

which targets a non-genic area on chromosome 8.

Based on TP53 loss along with the mutated gene

ERBB2 (another commonly mutated gene in

Gallbladder cancer), the mice appear to have

papillary Gallbladder cancer (Erlangga et al.2019).

The team proves that the activation of specific

genes combined with the loss of one or multiple

tumor suppressor gene can purposely control the

appearance of Gallbladder cancer in experimental

mice (Erlangga et al. 2019). Meanwhile, CRISPR-

Cas 9 has been applied in brain cancer. Similarly,

the researchers constructing four animal models

knock out Trp53, Nf1, Ptch1 and Pten that are

related to medulloblastomas with the assistance of

CRISPR-Cas9 technology. CRISPR-Cas9 featuring

higher accuracy and higher chance to succeed is

able to construct a GEMM model faster than

traditional one and therefore, it is applied in gene

knockout models of a variety of animals (Feng et al.

2024).

4 CURRENT CHALLENGES

BASED ON MEDICAL AND

ETHICAL LEVELS

Despite the superiority of CRISPR-Cas 9 technology,

there are remaining issues that need to be solved. The

off-target effect should be investigated when

CRISPR-Cas9 gene editing technique is put into

practice. In fact, off-target effect is a potential

problem in applications of many cancer treatments

and it is more frequent in human cells. The cause is

partially due to incomplete homologies between

gRNA and other regions of the genome and CRISPR-

Cas9 system binds to a sequence similar to the one

that should be cut (Rabaan et al. 2023). A mutation in

allele of off-target effect in a population will be

passed to the next generation based on genetic drift,

raising the number of offspring with this kind of

mutation. Unfortunately, off-target effect appears

more commonly in human cells. The form in which

CRISPR-Cas9 constituents are injected in cells is as

protein, DNA or RNA. In the simulation model of

mice, it is hard for the mice with genetic modification

to pass their changed genetic material to their

offsprings (Liu et al. 2023).

Another major consideration might be the

ethical aspects. To be more specific, gene editing

technology is seriously prohibited in the edition of

human reproductive cells. For example, “He Jiankui

affair”, a controversy over scientific progression of

gene editing technique and human ethical issues, is

a typical case. He Jiankui, a former professor and a

researcher in Southern University of Science and

Technology (SUS tech), China, created the first

genetically modified babies in human history. In the

year of 2018, He recruited 8 pairs of couples (eight

males positive for HIV antibodies) for the

experiment. Then, He manually edited the embryo’s

genes using CRISPR-Cas9 technology and had a

volunteer give birth to twins after artificial

insemination. He aimed to make the infants born to

be immune to AIDS by modifying their genetic

composition. It is studied that HIV would attach to

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the surface of white blood cell produced by CCR5

gene and the virus is unable to reach white blood

cells’ surface if proteins are generated by the

mutated type of CCR5 with the deletion of 32 base

pairs, thus people possessing mutated CCR5 gene

are immune to AIDS. Similarly, He and his

researching team used the CIRSPR-Cas 9 gene

scissor to cause 32 bp deletion to occur the CCR5

gene, intending to gain homologous mutation of it:

CCR5Δ32. On the contrary, this presents both huge

ethical problems and medicine ones. From the

medical perspective, it is true that CCR5 is an

essential part of the virus invasion, but there are

other ways of invasions, which have yet been

interrupted by any gene technologies, let along the

final outcome of this experiment was that the

newborns did not successfully gained CCR5Δ32.

As for ethical consideration, gene editing of human

embryos might raise social issues: the criteria of the

access to this technique and differentiation of the

whole society once CRISPR-Cas9 serves as a tool

to enhance human intelligence and physical ability

(Rabaan et al. 2023, Liu et al. 2023).

5 COMBINATION THERAPY:

CRISPR-CAS9 GENE EDITING

AND CAR-T CELL

IMMUNOTHERAPY IN

CANCER TREATMENT

Scientists are combining gene technology with other

existing immunotherapies to enhance immune cell

function, overcome tumor resistance and improve

treatment effect. The use of CRISPR-Cas9 along with

CAR-T cells therapy has been applied in medicine

(Razeghian et al. 2021). CAR-T cell (chimeric

antigen receptor) is genetically modified T-cells that

are capable of locating and destroying cancer cells. T-

cells separated from the plasma go through genetic

engineering and after that, T-cells with chimeric

antigen receptors (specialized proteins) on their

surface are now able to bind to antigens of tumor cells

and kill them (Akhoundi et al. 2021). Due to cell

division and growth, the number of these revamped

immune cells increases significantly to hundreds of

millions, which are injected back to the patient’s

body. Within a human body, CAR-T cells keep

proliferating, attacking and eliminating cancer cells

having distinguishable antigens on surface. However,

there are limitations leading to its small range of

application to patients, from high costs to difficulties

in gathering qualified T-cells, which urges doctors to

come up with a universal revamped CAR-T cell

(Akhoundi et al. 2021). To successfully be used in

clinics, universal CAR-T cell needs to pass from two

major barriers: self-immune rejective response and

increased safety after injection. Through

investigation and study, researchers found that when

identifying foreign antigens, TCRs (T-cell receptors)

may cause GVHD--graft versus host disease

happening after an allogeneic transplant. What is

more, human leukocyte antigen that locates on

allogeneic CART-cells activates immune

mechanisms preventing CAR-T cells from acting on

human bodies. That is what the CRISPR-Cas9 gene

engineering plays a part: using CRISPR-Cas9 to

knock out T-cell receptorβ chain and fundamental

components of HLA molecule--β-2-microglobulin

will silence the expression of TCRs and HLA

molecules (Akhoundi et al. 2021, Razeghian et al.

2021).

Experiments show CAR-T cells do not trigger

GVHD and function properly. Additionally, gene

editing excludes the influence of inhibitory signals

produced by immune check points. Cancer cells

sometimes utilize immune checkpoints to protect

themselves from being attacked. Once immune

checkpoint inhibitory molecules, PD-1, bind to its

receptors, PD-L1/PD-L2, the complex would reduce

CD8+T cell toxicity. An electroporation method is

adopted to knock out PD-1 in T-cells from Cas9

plasmids and sgRNA, deleting PD-L1 indirectly. In

the clinical level, the Cas9/sgRNA system precisely

inserts the α-PD-1 box into the GAPDH site of B

lymphocytes (Razeghian et al. 2021). Astonishingly,

the genetically edited B lymphocytes differentiate

into characteristic long-lived plasma cells (LLPCs)

both in an in-vitro setting and in in-vivo mice models.

These resultant LLPCs have the capacity to

constantly secrete novel antibodies. In xenograft

tumor mouse models, these antibodies can inhibit the

growth of human melanoma through an antibody-

mediated checkpoint-blocking mechanism. The

reduction of PD-L1 expression is carried out by

having CRISPR-Cas9 knock out Cdk5 gene (allows

PD-L1 expression) (Akhoundi et al. 2021, Razeghian

et al. 2021). Therefore, CRISPR-Cas9’s capability to

block PD-1 and PD-L1 will facilitate antitumor

responses and strengthen the ability of CAR-T cell

immunotherapy in cancer treatment.

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6 ADVANCEMENTS AND

RECENT DEVELOPMENTS IN

CRISPR-CAS9 GENE EDITING

TECHNOLOGY

As the investigation and experiments continue

progressing, more advancements have been achieved.

CRISPR-Cas9 is currently countering the problem of

targeting effect (as mentioned in part 3: Current

Challenges based on medical and ethical level) that

brings about undesirable genetic change when Cas9

scissors unexpected DNA sites (Rabaan et al. 2023).

Sending Cas9 nuclease to target cells remains

challenging, so designing delivery systems is what

people are doing recently. Chinese scholars develop

“a lactose-derived CRISPR-Cas9 delivery system”

that shows potential strengths in clinical trials.

Compared with viral delivery route, non-viral vectors

possess lower toxicity to human bodies (Alaa 2024).

Lactose, a type of disaccharide, is composed of one

glucose and galactose and its targeting feature

revealed from hepatic cancer cells can effectively be

used to treat liver diseases. Through one-pot ring-

opening reaction, LBP (lactose-derived branched

cationic biopolymer) with many disulfide linkages

and hydroxyl groups are synthesized. The physical

properties are studied to figure out the likelihood of

LBP as potential genetic vector and then, the toxicity

and targeting capability is tested in human hepatic

cancer cells (Alaa 2024). Scientists used BIRC5

(Baculoviral IAP Repeat Containing 5 that codes for

survivin that inhibits apoptosis of cells and regulates

mitosis) as targeting gene. Survivin expresses in

cancers but it cannot be found in normal cells. LBP’s

ability of targeting is seen when LBP delivers pCas9-

survivin (CRISPR-Cas9 plasmid for knocking out

survivin gene) to the mice HCC (hepatocellular

cancer cell) models (Liu et al. 2023, Alaa 2024).

7 LATEST DEVELOPMENTS IN

CRISPR-CAS9 GENE EDITING

TECHNOLOGY

Investigations and innovations of CRISPR-Cas9 gene

engineering technology made by scientists enable it

to progress in a rapid rate. Advancements in CRISPR-

Cas9 also provide hopes for patients who are in the

need of gene editing therapy for their cancer.

Researchers have been solving the problem of “off

targeting”: one of the biggest challenges faced by this

gene technique and therefore, it is necessary to select

proper delivery system (Alaa 2024). In 2024, a

delivery system with high efficiency, which is based

on nanoparticles is discovered. Consisting of cationic

lipids or lipid-like materials, nanoparticles are

protected from degrading and they can be transported

to tarted cells more efficiently and effectively.

Scholars are still working on enhancing its stability

and effects through experiments and research (Alaa

2024). CRISPR-Cas9 also demonstrates its wonderful

ability in gene modification of a variety of diseases.

In sickle cell anemia caused by the mutate gene HBB,

CRISPR-Cas9 is able to accurately modify HBB gene

and correct the disorder of gene compositions.

Furthermore, it succeeds in correcting several gene

mutations related to retinitis pigmentosa

(characterized by vision lost) (Liu et al.2023).

8 CONCLUSION

From the first discovery of “genetic scissor” in

Escherichia coli to the application in cancer treatment

and other diseases, it took human seven years. In

seven years of hard work and tons of trials, people in

this field figured out what the components in bacteria

are, uncovered the underlying mechanisms about how

CIRSPR-Cas9 works, began putting it into practice as

a method in clinical level, strived to solve the

problems present in it, combined gene therapy with

different immunotherapy to maximize its benefits and

strengths and finally, continue advancing CRISPR-

Cas9 system. Thanks to all of the researchers who

have ever stopped discovering things behind this

technique and persisted to improve it as much as they

can, people now have various information of different

aspects of CRISPR-Cas9 system on hands. It

originates from the acquired immune system of

bacteria and archaea as part of the resistant

mechanisms when foreign viruses invade the bacteria.

CRISPR- associated proteins (cas-9) and guide RNA

(gRNA) are the two major components of CRISPR-

Cas9 system. Cas9 is a nuclease, a protein capable of

cutting stranded DNA, causing DSB. gRNA, which is

a complementary to the target DNA sequence, will

guide the Cas9 protein to a specific location in the

genome.

As the gRNA binds to the Cas9 protein to form a

complex, it searches for the DNA sequence in the

genome that is complementary to the gRNA so that

Cas9 protein cuts the targeted DNA. Since DNA

breaks up, this system has two ways of repairments:

NHEJ and HDR, which have both advantages but

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drawbacks. The basic working principles enable

CRISPR-Cas9 to cut diverse segments of DNA under

changing circumstances. The efficiency, cheapness,

operational simplicity and high accuracy render gene

editing technology a rival therapy to treat cancers.

The majority of causes in cancer would be the

mutation of gene, which means that by studying

which gene may be responsible for stimulating the

growth and proliferation of cancer cells, scientists can

knock out the particular genes via gene tools.

However, the limitations and challenges in CRISPR-

Cas9 should not be ignored: off-target effect and

ethical issues. People are proposing solutions and

passing laws that strictly ban the use of gene editing

in human reproduction. CRISPR-Cas9 is a promising

treatment for cancer, offering patients and doctors

hopes. It is expected that scientists will have a

breakthrough in it one day and succeed in curing more

cancer patients.

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