Research and Application of CRISPR/Cas9 Technology in Oncology

and Blood Diseases

Hengyi Zhang

Guiyang No.1 Middle School, Guiyang, 550000, China

Keywords: CRISPR/Cas9, Tumor, Thalassemia, Hemophilia.

Abstract: The CRISPR/Cas system has been widely studied and applied to various fields as an efficient and simple gene

editing tool. So far, CRISPR/Cas9 systems have evolved from the initial Cas9 to a variety of systems such as

12a and 13a; and also from the initial targeting of DNA and RNA to transcriptional regulation and DNA

recycling. In addition, with the recognition of target sequences, certain CRISPR/Cas systems, such as Cas12,

Cas13, and Cas14 proteins, exhibit non-specific cleavage activity on other single-stranded DNA or RNA

molecules indirectly after the recognition of target sequences to initiate targeted cleavage activity under the

guidance of guide RNA. This paper introduces the principle of CRISPR/Cas9 and discusses its application in

oncology and hematological diseases, laying the foundation for the application of CRISPR/Cas9 technology

in diseases, which also shows that CRISPR/Cas9 has a vast prospect of development.

1 INTRODUCTION

Some diseases are so specific that the virus is present

in a baby's body at birth, and treatment becomes a

major problem. Even if cured in the body, if it is

present in the genes or not completely eradicated, it is

bound to recur or get worse later, and even future

generations may be affected. Thanks to the efforts of

many scientists around the world, gene editing

systems were introduced in the 1980s, an emerging

genetic engineering technique or process capable of

modifying specific target genes in an organism's

genome, and are now widely used in the study of

diseases such as tumors and blood disorders. 2020,

French scientist Emmanuel Carpentier and American

scientist Jennifer Doudna researched the "CRISPR

gene editing technology" was even awarded the

Nobel Prize in Chemistry. However, gene editing

technology involves random homologous

recombination of cells, and the efficiency of

recombination is extremely low (one in a million),

thus limiting the widespread use of gene technology.

The CRISPR/Cas system based on RNA-guided

recognition of DNA has the advantages of

simultaneous editing of multiple sites, simple

operational design, and ease of operation. The main

feature is the recognition and specific degradation of

invaded exogenous DNA, and the variety of these

DNAs. The CRISPR/Cas9 system, a key element of

the type II CAS proteins, has enabled scientists to

efficiently and precisely modify DNA or insert

substitutions, allowing for the rapid and efficient

construction of microbial models. The tremendous

developments achieved by the CRISPR system

provide important opportunities for treating genetic

diseases and designing desirable genetic traits, as well

as new methods for imaging living cells, etc., laying

the foundation for the next applications of gene

editing technologies and new discoveries.

Tumor development is accompanied by a

combination of multiple genes in multiple ways and

processes that are continuous and slow (

Liu, 2015

Both different genes at the same stage of development

and the same gene at different stages of development

play different biological roles, so research on tumors

requires interference with different gene expression

in the same cell at different times or in different cells

at the same time. To achieve this goal, rapid and

effective gene editing technology is needed to

artificially control gene expression, and the newly

developed CRISPR gene editing technology can

better fill this gap and more rapidly advance the

research in many aspects.

The occurrence of blood disorders is mostly

associated with genetic mutations. Gene editing

technology, makes it possible to cure genetic

mutation diseases. It is mainly a recombination

204

Zhang, H.

Research and Application of CRISPR/Cas9 Technology in Oncology and Blood Diseases.

DOI: 10.5220/0012018400003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 204-208

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

pathway triggered by exogenous introduction of

chromosomes with internal homologous

recombination through nucleotides in the absence of

nucleases, coupled with the use of viral genes as

templates for editing. However, because of the low

correction probability of conventional gene editing

techniques, it is clinically limited, and the

CRISPR/Cas9 system can effectively remedy this

aspect. This paper will address the principles of

CRISPR/Cas9 and its applications in cancer and

hematological diseases, laying the foundation for the

application of CRISPR/Cas9 technology in diseases,

which is seen to have a vast future.

2 PRINCIPLE OF CRISPR/CAS9

SYSTEM

The CRISPR/Cas9 system mainly consists of Cas9

protein, crRNA and tracrRNA, while the first three

were uniformly replaced by sgRNA as research

progressed (sgRNA consists of base complementary

pairing region, Cas9 handle and terminator) (Li,

2017). The adaptive immune system of bacteria and

archaea for defense against foreign invading viruses

and phages is the CRISPR/Cas9 Cas9 origin (Li,

2018). As an adaptive immune system, the RNA of

CRISPR directs Cas proteins to recognize invading

exogenous genomes in the form of base

complementation and to shear exogenous DNA (Liu,

2015). The protein sequence structures can be

classified into three types based on the difference in

the sequence structure of Cas proteins and are noted

as: type I, type II, and type III. Because type II

contains only Cas9 protein and type I and III have

multiple protein sequences, this section focuses on

type II (Liu, 2015). Cas9 nucleic acid endonuclease

has a large structural domain, in which the RuvC

nucleic acid endonuclease-like domain at the N-

terminus and the HNH nuclease-like activity in the

middle play an important role in cutting DNA. When

the Cas9 protein cleaves the DNA double strand

resulting in a broken cut, HNH shears the

complementary strand while RuvC cuts the non-

complementary strand. And the binding of the

CRISPR/Cas9 complex to exogenous spacer

sequences is not affected when both endonuclease

sites are mutated (Liu, 2015).

The CRISPR/Cas9 system is simplified and

consists of two parts: Cas9 protein and sgRNA. The

principles are as follows: 1. The successfully

expressed sgRNA forms a complex through its own

Cas9 handle and Cas9 protein; 2. The sequence of the

base complementary pairing region of the complex

sgRNA pairs with the target sequence of the target

gene through the principle of base complementary

pairing; 3. Cas9 uses its own nucleic acid

endonuclease activity to cleave the target DNA

sequence, as shown in Figure 1:

Figure 1: Cas9 cleaved DNA sequence (Li, 2017).

Cas9 proteins have multiple functions and large

molecular weight, SpCas9 and SaCas9 are now the

main widely used Cas9 proteins that have been

applied to Streptococcus pyogenes and

Staphylococcus aureus (Pan, 2020). In the type II

system, crRNA becomes mature crRNA under the

action of RNaseIII, followed by complementation

with the homologous repeat sequence in tracerRNA

hybridized into a double-stranded RNA dimer

structure, which then binds to the Cas9 protein to

form a cleavage complex, the targeting of which can

be specialized for gene editing such as knockdown,

insertion and targeted mutation of exogenous genes

(Pan, 2020). The second part of CRIPS/Cas9 is a non-

protein-coding RNA, tracrRNA, which is used to

complete the maturation of crRNA and subsequent

DNA shearing. In the case of Streptococcus

pyogenes, for example, this RNA is transcribed from

two initiation sites to produce a 171 nucleotide and 89

nucleotide sequence precursor tracrRNA of length,

respectively, both of which are then further processed

into a 75 nucleotide sequence mature tracrRNA, the

precursor portion of which can complement the

crRNA precursor, thereby facilitating its maturation

(Li, 2018).

The cleavage of Cas9 produces DSBs (double-

strand breaks) in eukaryotic cells by two repair

pathways: non-homologous end joining and

qualitative homologous repair (Li, 2017). In many

bacteria without the NHEJ (nonhomologous ending

joining) pathway, DSBs can also be repaired using a

Research and Application of CRISPR/Cas9 Technology in Oncology and Blood Diseases

205

cellular homologous recombination system with a

chromosome or plasmid-borne template However,

the process often results in arbitrary nucleotide

insertions or deletions near the cleavage site. (Li,

2017) Thus, the NHEJ pathway typically alters the

reading frame of the target gene cleaved by Cas9,

prompting a shift in the target gene sequence and

triggering the premature appearance of a stop codon,

leading to the previously mentioned knockout (Li,

2017). The HDR pathway allows for precise Cas9

protein editing of the cleavage target, with specific

nucleotide sequence editing, insertion, deletion and

substitution of specific nucleotide sequences.

However, since an efficient NHEJ pathway does not

exist in many bacterial genomes, the DBS formed by

Cas9 cleavage leads to cell death (Li, 2017).

3 CRISPR/CAS9 GENE EDITING

TECHNOLOGY IN

ONCOLOGY

Malignant tumor muscle, which can also be cancer,

refers to a disease caused by abnormalities of cells

These proliferating cells also invade other healthy

parts of the body, resulting in a malfunction of the

mechanisms that control cell division and

proliferation.

Treatment of cancer is variable depending on

many factors, including the type, location and amount

of disease as well as the health status of the patient.

Most treatments kill/remove cancer cells directly or

cause their eventual death by depriving them of the

signals needed for survival. Traditional treatments

include: radiation therapy, surgery, and systemic

therapy (chemotherapy). While radiation therapy is

relatively safe (no anesthesia required) and can kill a

large number of even invisible tumor cells in a

specific area, it is prone to post-cure wound

complications and poor healing; surgery has the

ability to remove all cancer cells in a small area, but

cannot kill microscopic lesions at the edge of the

tumor; chemotherapy has the ability to kill cancer

cells throughout the body, but cannot kill the tumor

alone as well as systemic Toxicity makes this

treatment option not the best choice either.

With the rapid development of high-throughput

measurement technology and biological information

technology, researchers have obtained a large amount

of genetic information in tumor cells. In the process

of tumor development, different genes play different

roles at the same stage or the same gene at different

stages (Liu, 2015). Therefore, studies related to tumor

gene function need to effectively interfere with

different gene expression at different stages of cell

differentiation. Therefore, the study of tumor gene

function requires effective interference with the

expression of different genes at different stages of cell

differentiation. On this basis, the effect of the gene on

tumor development should be investigated so as to

artificially and effectively control the level of gene

expression within the cell. CRISPR/Cas9 is currently

being investigated for three applications: 1. targeted

editing of target genes using this gene editing

technology, which has been widely used in genetic

engineering of eukaryotes and prokaryotes; 2.

genome-scale editing based on this technology,

coupled with high-quality sequential technology

screening in combination with phenotypic gene-

related technologies; 3. Use of Cas9 (dCas9) after

inactivation of nuclease activity to transform it into a

device that uses RNA guidance to develop a wider

range of uses by fusing effectors with dCas9 (Liu,

2015).

In 2014, Torres et al. first initiated the study of the

CRIPR/Cas9 technique to construct a muscle model

of malignancy. cas9, guided by specific sgRNA,

cleaves outside the site-specific DNA, causing

inversions and ectopics in the chromosome where the

cleaved DNA is located, thus accurately mimicking

the formation of some tumors such as Ewing's

sarcoma (Qu, Li, Jiang, etc. 2015). In the same year,

Xue et al. used CRIPS/Cas9 technology to

successfully suppress double mutations in two

oncogenes (p53 and pten), and animal liver cancer

models were constructed. (Qu, Li, Jiang, etc. 2015)

Platt et al. published a mouse tumor model in which

a DNA plasmid expressing Cas9 nucleic acid

endonuclease and sgRNA was injected into the liver

of mice using a hydraulic tail vein injection

technique; the Pten and P53 oncogenes were also

edited in mice (Liu, 2015), and the targeting AVV

subtype vector was designated as a CRISPR/Cas9

delivery system, allowing Cas9 to be specifically

expressed in the liver and lung. The mouse model of

lung adenocarcinoma was successfully constructed

(Qu, Li, Jiang, etc. 2015). In addition, a study on the

relationship between rectal cancer and the PIK3R1

gene reports the application of functional studies of

solid tumor-related genes. The researchers used

CRISPR/Cas9 technology to knock down the PIK3R1

gene at the level of rectal cancer cell lines, and later

examined the changes in interepithelial

stromalization, proliferation, and stem cell properties

of tumor cells in the knocked-down cells and wild-

type cell lines, respectively, thereby demonstrating

that the PIK3R1 gene has the function of regulating

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invasion, metastasis, and proliferation of rectal cancer

cells (Liu, 2015).

4 CRISPR/CAS9 GENE EDITING

TECHNOLOGY IN

HEMATOLOGICAL DISEASES

Most of the hematological diseases are associated

with genetic mutations and have limited and

ineffective therapeutic techniques, while the

application of gene editing technologies has made

another possibility for the treatment of hematological

diseases. Zinc finger ribonucleases and transcription

activator-like effector nucleases were found to be less

easy to design and less specific, as well as cytotoxic,

making these two techniques not widely available.

CRISPR/Cas9 is widely used in the field of disease

research because of its simplicity, targeting

specificity and affordability. In this section, we

present the progress of CRISPR/Cas9 technology in

two hematological diseases, thalassemia and

hemophilia (Li, 2018).

Thalassemia results in impaired hemoglobin

synthesis with symptoms similar to those of anemia

1. Fatigue and pallor due to low red blood cell

hematocrit, as well as skeletal disorders,

splenomegaly, and yellow fever. β - Thalassemia is

caused by mutations or small fragment deletions of

HBB (human bead protein) on chromosome 11,

which affects the transcription, shearing, and

translation of MRNA, resulting in β-hemoglobin

deficiency 1. This results in a deficiency of β-

hemoglobin.1 An abnormal excess of cells results in

an excess of α-protein chains and in damage to the

cell membranes of red blood cells, which may form

toxic aggregates if the damage is too great. The only

treatment currently available is hematopoietic stem

cell transplantation, but this is expensive and difficult

to match.1 In a 2012 clinical study in the United

States, researchers transfected β-thalassemia patients

with autologous CD34+ hematopoietic cells using a

wild-type β-globin transgenic TNS9.3.55 vector

(chronic viral vector), but no genetic markers were

detected in any of the four subjects.1 In recent years,

Liu et al. In recent years, Liu et al. used the

CRISPR/Cas9 system to screen for optimal gRNA as

well as a small molecule compound (L755507), in

combination with ssODN, to transfect iPSCs from β -

thalassemia patients, thereby repairing the deletion

mutation in β 41-42 (TCTT).1 Unlike the former,

Mettananda et al. used the α and β two-bead

imbalance protein chains resulting in ineffective

erythropoiesis and hemolysis in β -thalassemia

principle, greatly reduced the excess free α -bead

protein, thus greatly alleviating the clinical

manifestations of the patient.1 In addition, they used

the CRISPR/Cas9 system to delete mutations in the α

-bead protein MCS-R2 enhancer on the patient's

human hematopoietic stem cells to form α -

thalassemia.1 After editing, CD34+ cells

differentiated into mature erythrocytes, and the

reduction of α-hemoglobin corrected the

physiological imbalance between the two, thus

effectively improving the patient's symptoms (Li,

2018).

Hemophilia is an inherited bleeding disorder

associated with the X chromosome, which manifests

itself by patients bleeding longer and bruising easily

after surgery, along with an increased chance of

bruising and brain bleeding. Due to the difference in

genetic mutations, it can be divided into hemophilia

A and hemophilia B. Both have the same symptoms

with joint, muscle, and deep tissue bleeding.

Replacement therapy is currently the mainstay, but

the high cost of treatment and the production of

antibodies to clotting factors by frequent intravenous

infusions of clotting factor concentrates prevent

replacement therapy from being a long-term applied

and effective treatment.1 Hemophilia A is caused by

mutations in the F8 gene that encodes clotting factor

VIII. Researchers have induced pluripotent stem cells

(iPSC) from patients containing inversion mutations

in which the inversion gene was repaired to a normal

genotype using the CRISPR/Cas9 system and no off-

target mutations occurred throughout.1 Hemophilia B

is caused by mutations in the clotting factor IX (F9)

gene. The only current treatment is infusion of

clotting factor concentrate, which has a short half-life

and requires prolonged infusion. in 2011, researchers

at a children's research hospital and the University of

London, used a diphasic adeno-associated virus

(scAAV) carrying the optimized codon FIX gene for

treatment. After one year, stable expression of

activated IX factors was detected in all six subjects,

and the next 7-10 weeks, all showed specific T-

lymphocyte immune responses induced by the viral

capsid, accompanied by elevated transaminases.

Factors such as hepatotoxicity, reduction or loss of

introduced genes, and unmet demand for vector

production limited clinical application.1 Ohmori et al.

first abandoned conventional gene therapy by

injecting adenoviral vectors expressing Streptococcus

pyogenes Cas9 mRNA (Streptococcus pyogenes

Cas9) and sgRNA targeting exon 8 of the F9 gene in

mice8 (AVV8) was injected into the liver of wild

adult mice. A double-stranded DNA break occurred

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at the target site of the F9 gene, and the introduction

of homologous recombination repair (HDR) at this

site sufficiently enhanced FIX activity. In addition,

insertion of F9cDNA in introns and repair with NHEJ

or HDR was effective in restoring coagulation

function (Li, 2018).

Currently, gene editing technology is not mature

enough to be fully applied in the clinical setting.

However, studies have shown that CRISPR/Cas9

gene editing technology will become an effective

solution for gene therapy for the treatment of

hematological diseases.

5 CONCLUSION

CRISPR/Cas9 systems are widely used in life

sciences, agriculture, medicine, and industry because

of their simple design and ease of operation.

Scientists have used gene editing to construct animal

disease models to study a variety of difficult clinical

conditions. In addition, in agriculture, CRISPR/Cas9

has also accelerated genetic breeding in plants and

animals, etc (Wang, 2017).

With the continuous improvement of

CRISPR/Cas9 technology system, its application will

be expanded in the fields of energy, environmental

protection, and health. The technology can also be

combined with other types of technologies, such as

gene sequencing, gene expression analysis, disease

modeling, and drug delivery, thus making the

application of various technologies more extensive.

In addition to the construction of animal models, it

has been shown that CRISPR can also be involved in

the regulation of bacterial metabolism. In addition,

CRISPR technology is also widely used in other

fields, for example, editing the mosquito genome to

obtain specific antibodies to control the transmission

of Plasmodium. The application of improving the

growth rate and temperature resistance of fish, the

research and application of pet size and coat color

formulation, etc. also enable CRISPR technology to

be widely used in daily life (Wang, 2107).

Despite the rapid development of CRISPR

technology, there are still some problems. Scientists

are refining this technology by modifying the editing

proteins, using direct homologous enzymes, using

material assistance, and other measures to make

CRISPR/Cas9 technology applicable to more fields.

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