CRISPR‑Cas9 System for of Type 1 Diabetes Treatment

Ziqing Yang

Guangdong Country Garden School, Foshan City, Guangdong Province, China

Keywords: CRISPR‑Cas9, Type 1 Diabetes, Gene Therapy.

Abstract: Type 1 diabetes (T1D) is one of serious chronic metabolic illnesses, the reason behind is the hyposecretion of

pancreatic β cells because of the autoimmune system, and traditional therapy mainly focuses on insulin

replacement, which can not reverse the disease process. In recent years, the discovery and rapid development

of gene editing technology like the CRISPR/Cas9 system has provided a new idea for the radical treatment of

T1D. In this paper, the use of CRISPR/Cas9 technology for T1D treatment is systematically reviewed. First,

based on the pathogenesis of T1D, the core links of immune disorder and β cell function loss are described.

Secondly, we reviewed the development of CRISPR/Cas9 from a bacterial adaptive immune system to an

efficient gene editing tool, and analyzed its targeted DNA cutting and repair mechanism guided by sgRNA.

Further, two main application directions of the technique in T1D were discussed: editing immune cells to

inhibit autoimmune attack, and inducing stem cells to differentiate into functional β cells or enhancing

endogenous β cell regeneration. Current challenges, including off-target effects, low delivery efficiency, and

immunogenicity, are analyzed, and some optimization strategies. Finally, we look forward to the future

research direction, emphasizing the importance of interdisciplinary research in promoting clinical

transformation and providing ideas for future research.

1 INTRODUCTION

Diabetes mellitus is one of serious metabolic

illnesses, whose pathogenesis is the lack of insulin

and hyperglycemia as an external manifestation. The

diabetes mellitus is mainly divided into 2 types---type

1 diabetes and type 2 diabetes. Among them, the

treatment of type 1 diabetes is the most difficult. T1D

is an autoimmune disease caused by some relative

susceptibility genes leading to the error recognition of

T cells, they attack and damage the islet β cells then

contribute to the abnormal secretion of insulin. Of all

the diabetes patients around the world, T1D patients

account for about 5%~10% and increase at the speed

of 2%~3%. In the whole T1D patients, the major

group is children, the T1D patients under 20 years old

take up a percentage greater than 85% (Liang & Hu.,

2013). They all require lifelong medication therapy.

We have some current developments on treating

T1D. For example, insulin injection, which is the

most common therapy method, but it requires long-

term injection and there may be complications.

Patients can also take medicine, and they need to take

lifelong intake for treatment. The other methods are

immunotherapy and pancreatic islet transplantation.

However, immunotherapy has different effects on

different patients with complications, and it always

has a high price; pancreatic islet transplantation needs

high cost as well, and the donor sources are limited.

There are still no any complete cures at present.

Nevertheless, gene therapy as an emerging

treatment in recent years has the hope to completely

cure T1D. Gene editing technology can pinpoint a

specific spot in the genome and use specific nucleases

to achieve high profiling of the human genome’s

precise modification. CRISPR/Cas9 system has the

most potential among all kinds of gene editing

technology. Compared to most other gene editing

technologies, the CRISPR/Cas9 system only needs to

design 1 sgRNA to edit related genes, which has a

simpler operation, lower cost and higher efficiency.

CRISPR/Cas9 system can precisely modify stem cells

to achieve treatment for T1D. This article will

research the development of the CRISPR/Cas9

system in the treatment of type 1 diabetes.

218

Yang, Z.

CRISPR-Cas9 System for of Type 1 Diabetes Treatment.

DOI: 10.5220/0014464900004933

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 218-222

ISBN: 978-989-758-789-4

2 TYPE 1 DIABETES

2.1 Factors Cause Type 1 Diabetes

Type 1 diabetes is one type of diabetes mellitus and

belongs to the autoimmune disease. The core

mechanism is the attack of T cells (T lymphocytes) to

pancreatic β cells. There are a few factors that lead to

this mechanism: The first one is the hereditary factor,

some susceptibility genes related to T1D may

generate to the next generation, which is the

necessary basis for T1D. It affects immune

recognition, making an abnormal tendency of the

recognized function of the immune system and cells,

it may lead to the core mechanism above and increase

the risk of T1D. The second factor is the

environmental factor. Some environmental factors

like virus infection and chemical substances may also

increase the possibility of pancreatic β cells’

dysfunction by mistakenly attacking T cells, finally

raising the risk of T1D. The third factor is abnormal

autoimmunity regulation. It is the immediate cause of

T1D due to the interaction of hereditary genetic

factors and environmental factors, which directly

leads to T cells’ attack on pancreatic β cells.

2.2 The Detailed Mechanism behind

Under the influence of the above factors, T cells

attack β cells mainly through 2 ways respectively.

The first and also the core pathway is the direct killing

by the CD8⁺T cells which is one type of T

lymphocytes. CD8⁺T cells recognize an antigen on

the surface of pancreatic β cells, release perforin and

granzymes to form membrane pores and let the

granzymes enter the cell, activate the caspase

cascade, and induce β cell apoptosis (Atkinson et

al.,2011). The second pathway is the indirect killing

by the CD4⁺T cells. On one hand, CD4⁺T cells secret

chemical substances like IFN-γand TNF-α to activate

macrophage and aggravate the damage to pancreatic

β cells. On the other hand, CD4⁺T cells can also assist

B cells to produce the specific antibodies to damage

pancreatic β cells again (Chow et al., 2014). These

abnormal responses of T cells are the result of

abnormal gene expression.

2.3 The Difficulties of Treating T1D

According to the nosogenesis of T1D, the difficulty

of the treatment of T1D is that the constant attack of

the immune system is difficult to suppress

completely. There are a number of T cells’ attack

pathways working together to cause the damage of

pancreatic β cells. A single-target intervention may

not be able to stop the attack altogether. Also the

Existing therapies are easily destroyed by the

inflammatory microenvironment in the body and are

difficult to maintain long-term tolerance.

Additionally, Long-term immunosuppression carries

a risk of side effects, it can increase the risk of

infection, and tumor, and needs long-term

medication. However, using gene editing technology

like CRISPR/Cas9 system, can make a personalized

plan for each patient and achieve precision targeted

therapy. Furthermore, it can also promote β cell

regeneration and protection, and achieve the effect of

long-term treatment.

3 CRISPR/CAS9 SYSTEM

COMPOSITION AND

WORKING MECHANISM

CRISPR/Cas9 system is a simple and useful tool that

efficiently modifies endogenous genes in various

species and cell types (Hryhorowicz et al., 2017). It

enables targeted gene knockouts, base editing,

epigenetic modulation, and therapeutic applications

across eukaryotes. In 1987, Japanese scientist

Yoshizumi Ishino's team has for the first time

identified clusters of regularly spaced short

palindromic repeats (CRISPR) in the E. coli genome,

but the function is unknown (Ishino et al., 1987).

Until 2012, Jennifer Doudna, in collaboration with

Emmanuelle Charpentier, demonstrated that Cas9 can

cut specific DNA under sgRNA (single-guide RNA)

guidance, enabling CRISPR programmability for the

first time (Martin et al., 2012), and won the Nobel

Prize in Chemistry in 2020. At present, CRISPR/Cas9

technology was applied in multiple fields, including

but not limited to the treatment of diseases such as

cancer, agriculture and ecological applications.

3.1 Composition of the CRISPR/Cas9

CRISPR/Cas9 system is derived from an adaptive

immune mechanism to resist the invasion of foreign

genetic materials like bacteriophages. It consists of

Cas9 nuclease and single guide RNA (sgRNA). Cas9

nuclease comes from bacteria and archaea, it plays a

key role in recognizing and cutting both strains of

foreign DNA. sgRNA is an artificially engineered

RNA sequence from crRNA (CRISPR RNA) with

specific targeting at the 3 'end and tracrRNA (tans-

CRISPR-Cas9 System for of Type 1 Diabetes Treatment

219

acting CRISPR RNA) produced by trans-activated

ribonucleic acid gene expression at the 5' end.

As for the design and synthesis of sgRNA. To

design the sgRNA, first, Identify the target gene locus

you want to edit according to the protospacer adjacent

motif (PAM) sequence. Secondly, select an

appropriate length and scaffold sequence for a

specific secondary structure to make sure it can

accurately match with the target DNA sequence. Next

comes to the synthesis of sgRNA, there are 2 different

approaches. One is chemical synthesis: by chemical

synthesis technology, accurately controls the

sequence and modification of nucleotides, to produce

sgRNA, but it is difficult and low-yield (Hoy et al.,

2022). Another one is In vitro transcription synthesis:

the DNA template encoding sgRNA is synthesized

and then transcribed in vitro by RNA polymerase to

produce sgRNA. The design and synthesis of sgRNA

make sure the sgRNA will only bind to the target

sequence but not other regions of the genome.

3.2 Working Mechanism of the

CRISPR/Cas9 System

After having a specific sequence of sgRNA which is

complementary to the target mutant gene sequence

affecting pancreatic β cell in the genome, we extract

the multipotential stem cells from a patient and

introduce the CRISPR-Cas9 system into the extracted

stem cells (through carrier). The sgRNA binds to the

specific part of DNA, followed by Cas9 nuclease. The

Cas9 binds to the sgRNA and corresponding DNA,

and makes a cut across both strands of the DNA. The

cell recognizes that the DNA is damaged and tries to

repair it. It activates its own DNA repair mechanisms,

then we start to intervene.

As the figure 1 shown, the process to repair the

severed DNA also have 2 different methods. The first

one is non-homologous end joining (NHEJ), it means

when the double strains break, it quickly joins the two

ends of the broken DNA together, and in this process,

there is always some insertion or deletion of genes at

the junction part. Thus genes that enhance the

function of islet beta cells can be introduced in and

abnormal genes that impair the function of pancreatic

β cells can be knocked out. The second method is

homologous directed repair (HDR), it needs to offer

a foreign DNA template homologous to the sequence

at both ends of the broken DNA. When the cell starts

to repair, it will accurately integrate the genetic

information on the template into the break site to

achieve accurate gene editing-like replacement. It

repairs the mutated gene sequence to a normal one,

returning the pancreatic β cells’ function to normal.

After that, the edited stem cells are induced to

differentiate into pancreatic β cells and transplanted

back into the patient's body to return to the normal

function of the genes, thus promoting the normal

work of pancreatic β cells and synthesis of insulin.

(Lotfi et al., 2023)

Figure 1: Two Process of the CRISPR/Cas9 System’s

Mechanism (Lotfi et al., 2023).

4 CURRENTLY USED IN THE

TREATMENT OF T1D

The application of the CRISPR/Cas9 system in the

treatment of T1D is currently in the early stage of

research, focusing on gene-edited immune cells, islet

cell regeneration, and immune tolerance induction.

4.1 Suppress Autoimmune Responses

Luo’s team used the CRISPR/Cas9 system to knock

down the expression of co-stimulatory molecules on

the DCs surface. Cationic lipid-assisted PEG-b-

PLGA nanoparticles (CLAN) were used as the

delivery system and vector, then injected into the type

1 diabetes model mice, finally found that blood sugar

levels were significantly reduced in the treatment

group and damage to islet beta cells was reduced by

50 percent. It inhibits the ability to activate effector T

cells and suppress the autoimmune responses (Luo et

al., 2020). More innovatively, Martina S Hunt’s team

used CRISPR/Cas9 system through dual-locus, dual-

HDR editing to achieve double HDR synergism,

using the RNP complex formed by purified Cas9

protein and sgRNA. It enables Treg to obtain antigen-

specific recognition ability while enhancing the

expression stability of FOXP3, which prevents Treg

BEFS 2025 - International Conference on Biomedical Engineering and Food Science

220

from transforming into effector T cells in an

inflammatory environment. This directly inhibits the

activation of autoreactive T cells, thereby restoring

the immune system's tolerance to autoantigens. (Hunt

et al., 2023)

4.2 Regeneration of Pancreatic β Cells

In 2006, for the first time, Samson S L and Chan L’s

team systematically demonstrated the feasibility of

gene therapy PDX-1, NeuroD/BETA2 and

Neurogenin 3 to promote the regeneration and

functional reconstruction of β cells by

transdifferentiation of non-endocrine cells. (Samson

S. L. & Chan L. 2006). Although limited by the

technology of the time, it laid the theoretical

foundation for the breakthrough technology of

CRISPR/Cas9. After that, Saleh’s team reviews the

potential of pancreatic α cell to β cell

transdifferentiation in the treatment of diabetes

mellitus, focusing on the mechanism of

reprogramming α cells into insulin-secreting cells

induced by gene editing (such as overexpression of

PDX-1) or small molecule compounds. The effect of

improving blood glucose homeostasis in animal

models was verified. (Saleh et al., 2021)

5 CHALLENGS AND

PROSPECTS

5.1 Limitation of CRISPR/Cas9 System

The first limitation is the delivery challenge. Until

now, there are many ways to introduce the

CRISPR/Cas9 system into the cells. For example,

viral vectors like lentivirus and non-viral vectors like

plasmid vectors. However, all of them have their own

positive and negative aspect. We still need to find a

safer and more efficient method to deliver the

CRISPR/Cas9 system. Next the immunoreaction

challenge is also important. The injection of Cas9

nuclease as a foreign substance, it will activate the

immune reaction inside the human body, attacking

and destroying the Cas9 enzyme, also prevents the

gene editing and affects CRISPR/Cas9 system

effectiveness. The third one is an off-targeting

challenge. CRISPR/Cas9 has a high rate of off-

targeting. The sgRNA may bind with incorrect DNA

sequence and lead to gene editing errors with a high

rate of gene mutation. The fourth restriction is an

ethical challenge. There are still many ethical and

social concerns about using CRISPR/Cas9

technology to change the gene sequences in embryo

cells. The final one is a technical challenge. The

technology and machinery need to be very advanced.

In addition, facing different patients, we need to

consider changing situations and produce different

gRNA, which will contribute to a high cost of time

and money. We still need to find a cheap and more

accurate way to use the CRISPR/Cas9 system and

make sure everyone’s type 1 diabetes can be treated.

(Cheng et al., 2023)

5.2 Improvement

To deal with these limitations, we can search for new

delivery carriers to optimize the delivery system, or

attempt to deliver Cas9 mRNA instead of protein,

reducing immunogenicity and improving expression

efficiency and delivery efficiency. Try a double

sgRNA design. using two sgRNA to simultaneously

target both ends of the target region may improve

specificity. Try to scale and automate production, and

simplify the production process, reduce the

production cost. Let everyone afford this treatment.

6 CONCLUSION

To sum up, CRISPR/Cas9 technology for Type 1

Diabetes (T1D) treatment represents a

groundbreaking frontier in both gene editing and

diabetes research. This review has systematically

explored the pathogenesis of T1D, the historical

development and mechanistic principles of

CRISPR/Cas9, and its transformative potential in

addressing the challenges of T1D. Key findings

highlight the versatility of CRISPR/Cas9 in targeting

immune dysregulation, promoting β-cell

regeneration, and inducing immune tolerance,

offering hope for a curative approach to this chronic

autoimmune disease. Apart from its promise,

CRISPR/Cas9 still faces challenges such as off-target

effects, immunogenicity, and delivery efficiency. The

design of high-fidelity Cas9 variants, new delivery

systems, and immune escape strategies is gradually

addressing these issues. The research on the

CRISPR/Cas9 system for treating type 1 diabetes

treatment can show the potential of this technology in

treating type 1 diabetes and promoting the clinical

transformation. While significant progress has been

made, we still need to explore the way to improve

specificity and safety, optimize delivery strategy, and

more multi-disciplinary collaboration to try to reduce

CRISPR-Cas9 System for of Type 1 Diabetes Treatment

221

the cost of treatment, so that everyone can achieve the

treatment of disease.

REFERENCES

Atkinson, M. A. 2011. How does type 1 diabetes develop?:

the notion of homicide or β-cell suicide revisited.

Diabetes 60(5):1370-9.

Cheng, Y. 2023. The promise of CRISPR/Cas9 technology

in diabetes mellitus therapy: How gene editing is

revolutionizing diabetes research and treatment. J

Diabetes Complications 37(8):108524.

Chow, I. T. 2014. Assessment of CD4+ T cell responses to

glutamic acid decarboxylase 65 using DQ8 tetramers

reveals a pathogenic role of GAD65 121-140 and

GAD65 250-266 in T1D development. PLoS One

9(11):e112882.

Hoy, A. 2022. Bio-Orthogonal Chemistry Conjugation

Strategy Facilitates Investigation of N-

methyladenosine and Thiouridine Guide RNA

Modifications on CRISPR Activity. CRISPR J

5(6):787-798.

Hryhorowicz, M. 2017. CRISPR/Cas9 Immune System as

a Tool for Genome Engineering. Arch Immunol Ther

Exp (Warsz) 65(3):233-240.

Hunt, M. S. 2023. Dual-locus, dual-HDR editing permits

efficient generation of antigen-specific regulatory T

cells with robust suppressive activity. Mol Ther.

31(10):2872-2886.

Ishino, Y. 1987. Nucleotide sequence of the iap gene,

responsible for alkaline phosphatase isozyme

conversion in Escherichia coli, and identification of the

gene product. J Bacteriol169 5429-5433.1987

Liang, M. L. & Hu， Y. H. 2013.The progress of the

epidemiology of type 1 mellitus. Chinese journal of

disease control & prevention 17(04):349-353.

Lotfi, M. 2023. Application of CRISPR-Cas9 technology in

diabetes research. Diabet Med. 41(1):e15240.

Luo, Y. L. 2020. An All-in-One Nanomedicine Consisting

of CRISPR-Cas9 and an Autoantigen Peptide for

Restoring Specific Immune Tolerance. ACS Appl

Mater Interfaces 12(43):48259-48271

Martin, J. 2012. A Programmable Dual-RNA–Guided

DNA Endonuclease in Adaptive Bacterial Immunity.

Science 337, 816-821

Saleh, M. 2021 . Alpha-to-beta cell trans-differentiation for

treatment of diabetes. Biochem Soc Trans. 49(6):2539-

2548.

Samson, S. L. & Chan, L. 2006. Gene therapy for diabetes:

reinventing the islet. Trends Endocrinol Metab.

17(3):92-100.

BEFS 2025 - International Conference on Biomedical Engineering and Food Science

222