Diabetes is a chronic disease where the body cannot properly regulate blood sugar, either because the pancreas doesn’t produce enough insulin (Type 1) or the body can’t use insulin effectively (Type 2). Conventional treatments, like insulin injections or oral medications, manage symptoms but don’t fix the root cause, leaving patients dependent on lifelong therapy and at risk for complications like hypoglycemia and weight gain. Type 1 diabetes, an autoimmune disease where immune cells destroy insulin-producing beta cells, is influenced by genetics, especially HLA gene variations, and environmental factors. Worldwide, 537 million adults live with diabetes, and Type 1 is rising 3–5% annually, affecting over 1 million people under the age of 20. Emerging gene-editing technologies like CRISPR Cas9 show strong success in correcting disease-causing mutations, offering the potential to restore natural insulin production and even reduce susceptibility in future progeny, moving treatment from symptom management toward a potential cure.

For my research, after listing a few interests of mine, I began doing background reading to understand gene editing, focusing on non-viral vectors, RNA therapies, and CRISPR Cas9, since my original topic was Developing Precise CRISPR-Cas9 Therapy Strategies to Correct Hereditary Diabetes Mutations. I explored how non-viral plasmid vectors can deliver healthy genes into cells safely, how RNA therapies can supply instructions for missing proteins or silence harmful genes, and how CRISPR Cas9 can precisely cut and repair DNA at specific sites. After realizing that CRISPR Cas9 might be the best solution to hereditary diabetes treatment, I learned about key genes like TCF7L2, KCNJ11, and INS, and how their mutations disrupt insulin production. My research questions focused on how to prevent hereditary diabetes, how gene therapy can improve hereditary diseases, and how CRISPR could be delivered more safely and precisely. After presenting my initial proposal to the science department at my school and receiving feedback, I decided to narrow my scope because the project was too complex to complete fully, so I focused on comparing CRISPR treatment to conventional therapies. With guidance from my mentors, I learned to use PubMed and other scientific sources to find credible information. I researched CRISPR applications, primarily using review papers and collected data on conventional diabetes treatments. Then, I created a comparison table to evaluate their effectiveness relative to each other. Finally, I explored real world examples of CRISPR being used, analyzing how it has been applied to correct genetic mutations and improve outcomes in diseases like diabetes.

What is CRISPR Cas9? CRISPR Cas9 is a gene-editing tool that is able to cut DNA at a specific location using the Cas9 enzyme, often described as “molecular scissors”. The cell’s natural DNA repair mechanisms then repair the break, allowing targeted genetic modifications. It uses guide RNA (gRNA) to locate a specific DNA sequence, then uses the Cas9 enzyme to cut the DNA at that point, creating double-strand DNA breaks at specific target sites in a genome. This gene editing tool has successfully been employed as an ex vivo gene-editing tool in embryonic and patient derived stem cells. In addition, it enables researchers to further understand pancreatic beta-cell development, and function. Before CRISPR, TALENs were used to edit human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). They were also used to fix genetic defects in diseases like sickle cell, and to change T cells so they could resist drugs or fight tumors.  The use of RNA-guided nucleases allow for efficient testing of therapeutic methods in diabetes. Nucleases are enzymes that cut DNA or RNA at specific locations. Unlike older gene editing tools like Transcription Activator-Like Effector Nucleases (TALENs) or Zinc-Finger nucleases (ZFNs), CRISPR Cas9 is faster because because only the guide RNA needs to be changed to target a new gene, and it’s cheaper since making RNA is easier than designing new proteins every time. However, CRISPR Cas9 has a risk of off-target cuts, which can lead to unintended mutations or genetic alterations. The tool also raises ethical concerns over germline editing, meaning heritable changes. 

Method Development of CRISPR Cas9. CRISPR Cas9 evolved from the first introduction of genome modification by researchers Rudin and Haber. They used an endonuclease, which are the enzymes acting as "molecular scissors", to show the efficiency of gene targeting was increased by double-strand DNA breaks (DSB). Their study also investigated Homologous Recombination (HR), and how it was not practical in higher eukaryotic cells because the low efficiency led to unreliable DNA repair. To test this theory, researchers inserted specific DNA cleavage sites into the mouse genome so they could intentionally create double-strand breaks using the I-SceI enzyme. They found that mammalian cells mainly repaired these breaks through HR, which proved targeted genome modification was possible, although not very efficient. However, reprogramming these early nucleases was difficult, which led to the development of more flexible and efficient tools like CRISPR-Cas9.

CRISPR-Cas9 Gene Therapy for Sickle Cell Disease Sickle cell disease (SCD) is caused by a single mutation in the β-globin (HBB) gene, a vital protein-coding gene that provides instructions for making beta-globin, a critical component of hemoglobin used by red blood cells to transport oxygen throughout the body. It is a group of inherited blood disorders that cause red blood cells to become hard, sticky, and C-shaped (like a sickle). CRISPR can fix this mutation, however, electroporation, which is the electric shock delivery, damages the cells. Electroporation has a high editing efficiency, but the toxicity kills many stem cells in the process. Stem cells are undifferentiated cells that can develop into specialized cell types, and hematopoietic stem cells (HSCs) specifically produce all blood cells. In addition, using integrating lentiviruses to deliver CRISPR can cause long-term Cas9 expression, increasing the risk of unintended genetic damage, or genotoxicity. For these reasons, scientists are working to develop a non-toxic, non-integrating CRISPR delivery system that not only corrects the sickle cell mutation, but also avoids permanent DNA integration, and works efficiently in hematopoietic stem cells (HSCs), which are sensitive blood stem cells that are hard to collect.

Some of these new strategies include delivering the Cas9 protein directly instead of Cas9 DNA, using non-integrating lentiviral vectors (NILVs). In this method, Cas9 protein and guide RNA enter the cell and find the sickle mutation in the β-globin gene, and Cas9 makes a double-strand break at that location. Donor DNA with the correct β-globin sequence is also delivered, and the cell repairs the break using homologous recombination, which replaces the mutated sequence with the correct one. Experimental models, such as converting green fluorescent protein (GFP) into yellow fluorescent protein (YFP), showed high editing efficiency and confirmed that Cas9 activity was temporary. In sickle cell models, this method corrected up to about 40% of the β-globin protein, with minimal cell death and no detectable off-target effects. Researchers also found that Cas9 can be delivered without permanent integration, larger donor DNA improves repair efficiency, and NILVs are much less toxic than electroporation. These results show that this method can fix the root genetic cause of sickle cell disease while protecting stem cell health, making it a safer and more effective potential long-term treatment. This is also relevant to diabetes, because similar CRISPR delivery methods could be used to safely edit pancreatic beta cells or stem cells to restore insulin production, which shows that CRISPR could become a safer long-term treatment for diabetes.

Applications of CRISPR in Animal Models CRISPR-Cas9 has been used to create animal models of diabetes by knocking out genes involved in glucose regulation. In one study, rats were used as the chassis organism, and researchers knocked out the leptin receptor (Lepr) gene using CRISPR-Cas9. The leptin receptor is important for regulating body weight and insulin sensitivity. The purpose of this modification was to create a more accurate model of Type 2 Diabetes compared to older rodent models. After the gene knockout, the rats became obese, developed insulin resistance, and had persistent high blood sugar levels. Their glucose metabolism was impaired, similar to humans with Type 2 Diabetes. This showed that CRISPR-Cas9 successfully created a reliable disease model that could be used for studying diabetes and testing new treatments.

CRISPR-Cas9 was also used to create a large animal model using pigs as the chassis organism. Researchers knocked out the insulin (INS) gene, which is responsible for producing insulin, a hormone required for regulating blood glucose levels. The purpose of this modification was to create an animal model closer to human physiology for diabetes research and treatment testing. As a result, the pigs developed hyperglycemia (high blood sugar) and glucosuria (glucose in the urine), which are key symptoms of diabetes. Their glucose metabolism was severely impaired due to the lack of insulin. This demonstrated that CRISPR-Cas9 could successfully create large animal models of diabetes, which are important for studying disease progression and developing therapies such as insulin replacement and islet transplantation.  + using pigs to get insulin, 

Type 1 Diabetes Diagnosis In Canada, type 1 diabetes is diagnosed if a patient’s A1C, which is their average blood sugar levels over the past 2 - 3 months, is 6.5% or higher on two separate tests. A1C measures the average blood sugar over the past 2–3 months. Blood glucose is measured in mmol/L in Canada, with normal fasting levels between 4.0–5.5 mmol/L and diabetes diagnosed at 7.0 mmol/L or higher. Once diagnosed, treatment requires lifelong insulin therapy and regular blood sugar monitoring.

Glycemic Management in Adults Insulin has been used as a lifesaving pharmacological therapy that is primarily produced by recombinant DNA technology. This is made either structurally identical to human insulin, or as a human insulin modification (insulin analogues) to alter pharmacokinetics (the movement of drugs in the body). Human insulin and insulin analogues are preferred, and used by most adults with type 1 diabetes, although preparations of animal-sourced insulin are still accessible in Canada, though rarely needed. Insulin types are classified by duration, onset and peak, meaning how long the insulin works in the body, how quickly it starts to lower blood sugar, and when it has its strongest effect. Premixed insulin is often unsuitable, so basal-bolus injections, or insulin pumps (CSII) are preferred to maintain blood sugar while avoiding hypoglycemia.

People with type 1 diabetes begin insulin therapy immediately at diagnosis. Treatment requires choosing an insulin regimen and learning how to manage blood sugar through monitoring, diet, and lifestyle adjustments. The standard approach is a basal-bolus regimen or continuous subcutaneous insulin infusion (CSII), which attempts to mimic normal pancreatic insulin secretion. Basal insulin (long-acting) controls blood sugar between meals and overnight, while bolus insulin (rapid-acting) controls blood sugar after meals. Regimens are individualized based on age, health, lifestyle, and ability to self-manage. Many patients experience a temporary “honeymoon period” after diagnosis where insulin needs are lower, but this phase is short-lived and lifelong insulin dependence follows.

Although modern insulin analogues and insulin pumps have improved glucose control and reduced complications, hypoglycemia (dangerously low blood sugar) remains the most common and serious side effect. Intensive insulin therapy lowers the risk of long-term microvascular and cardiovascular complications, as shown in the DCCT trial, but it requires constant monitoring and carries ongoing risks. Technologies like continuous glucose monitoring (CGM) and sensor-augmented pumps improve A1C levels and reduce hypoglycemia, yet they do not cure the disease, they only manage it. This highlights the key limitation of conventional therapy: it replaces insulin but does not correct the underlying autoimmune destruction of pancreatic beta cells, which is where gene-editing approaches like CRISPR aim to intervene.

What is Type 1 Diabetes (T1D) Type 1 diabetes is an autoimmune destruction of insulin producing β cells in the pancreas, specifically islets of Langerhans, which are the hormone-producing cells. CD4+ and CD8+ T cells attack the β cells, and genetics, especially variations in HLA genes on chromosome 6, plus environmental factors, increase risk. The Eisenbarth model (1986) describes T1D as a multi-step disease, which means prevention or treatment could target different stages. T1D is increasing 3–5% per year, and over a million people under 20 live with it worldwide.

Current Treatment Therapies for Type 1 Diabetes Insulin Replacement Therapy: Losing insulin means lifelong insulin therapy. First used in 1922 with animal pancreas extracts, early treatments had inconsistent absorption and high hypoglycemia risk. Modern insulin analogues mimic natural insulin better, but still cannot fully replicate pancreatic function. Delivery includes multiple daily injections or continuous infusion via pumps. Limitations include hypoglycemia risk, lifelong dependence, weight gain, and possible insulin resistance. New approaches like oral insulin devices (e.g., SOMA) are under study.

Artificial Pancreas (Closed-Loop Systems): Combines continuous glucose monitoring (CGM) with an insulin pump for automatic insulin delivery. Benefits include more precise dosing, better overall glucose control, and fewer hypoglycemic events, making daily management easier for patients. Immune Therapies: Since T1D is autoimmune, treatments aim to suppress or modify the immune response. Drugs studied include cyclosporin A, anti-CD3/CD20 antibodies, and IL-1 inhibitors, but most only temporarily preserve β cell function. New strategies involve regulatory T cells (Tregs) or nanoparticles targeting pancreas-specific immune responses, with ongoing research to improve effectiveness.

SGLT2 Inhibitors: These drugs block glucose reabsorption in the kidneys, lowering blood sugar by increasing glucose excretion in urine. They may help as adjunct therapy, but long-term safety in T1D is still being studied.

Peptide Hormone–Based Therapies: Gut hormones like GLP-1 help regulate glucose and insulin. GLP-1 receptor agonists reduce post-meal glucose spikes, decrease glucagon, and slow stomach emptying. Other peptides like oxyntomodulin (Oxm), GLP-2, and peptide YY (PYY) may protect β cells and improve insulin secretion, but long-term benefits are not fully known.

Xenotransplantation of Islets: Transplanting insulin-producing cells from animals, usually pigs, can address donor shortages. Challenges include immune rejection, infection risk, and ethical concerns. Genetically modified pigs are being developed to reduce rejection and improve safety.

Induced Pluripotent Stem Cells (iPSCs): Adult cells can be reprogrammed into stem cells using Yamanaka factors (Oct4, Sox2, c-Myc, Klf4). iPSCs are patient-specific, reducing rejection and avoiding most ethical issues. Limitations include complex production, tumor risk (teratomas), and high cost.

Many advanced treatments attempt to manage blood glucose or suppress the autoimmune response, but none permanently correct the underlying genetic and immune dysfunction.

My data compares conventional diabetes treatments with CRISPR-based treatments. Conventional treatments, like insulin injections, artificial pancreas systems, and xenotransplantation, help manage blood sugar but come with daily routines, risks like low blood sugar, and high costs. CRISPR treatments, TALENs, and ZFNs aim to fix the root cause of the disease by editing genes, which could reduce or even eliminate the need for daily insulin. While CRISPR is still experimental and expensive, it offers more precise treatment, fewer long-term inconveniences, and the potential for permanent solutions. Overall, the data shows that conventional treatments are effective for managing symptoms now, but CRISPR has the potential to transform how diabetes is treated in the future.

CRISPR is truly amazing because it targets the root cause of diseases like Type 1 diabetes instead of simply managing symptoms. Unlike insulin therapy, which requires daily injections and careful monitoring, CRISPR has the potential to permanently restore natural insulin production by correcting faulty genes in a patient’s own cells. A powerful example of CRISPR in action is a patient-specific in vivo treatment for CPS1 deficiency, a rare metabolic disorder that prevents ammonia removal and can be fatal. Instead of just managing symptoms with diet, drugs, or liver transplant, researchers corrected the faulty CPS1 gene in liver cells using base editing, which changes a single DNA letter without cutting both strands, making it safer. The therapy, delivered using lipid nanoparticles with a custom gRNA and mRNA instructions, is temporary and non-integrating, reducing long-term risk. After extensive testing in cells, mice, and primates, it was given to a 7-month-old infant, who showed lower ammonia, improved protein tolerance, reduced medication, and better growth, with no serious side effects. While long-term follow-up is needed, this case proves that personalized CRISPR therapies can be safely developed and could potentially treat other genetic diseases, including hereditary diabetes.

Type 1 diabetes cases continue to rise worldwide, and while insulin therapy remains the main treatment, it does not cure the disease. Promising strategies for the future include artificial pancreas systems, stem cell–derived beta cells, encapsulation devices, xenotransplantation, and combination therapies. Together, these approaches aim to restore insulin production and regulation, with the ultimate goal of achieving long-term insulin independence for patients.

While insulin therapy remains a temporary treatment, the emerging field of gene editing, specifically with the CRISPR Cas9 should continue to be researched and studied, because of the strong success rate of it, and potential that would permanently change the healthcare system.

(n.d.). https://pmc.ncbi.nlm.nih.gov/articles/PMC12033450/#sec2-14791641251334726 (n.d.). https://pmc.ncbi.nlm.nih.gov/articles/PMC5815912/ (n.d.). https://pmc.ncbi.nlm.nih.gov/articles/PMC6058482/ The art of vector engineering: towards the construction of next‐generation genetic tools. (n.d.). PMC. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC6302727/ Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease. (2021). Retrieved February, 2026, from https://www.cell.com/molecular-therapy-family/advances/fulltext/S2329-0501%2821%2900037-1 Challenges in delivery systems for CRISPR-based genome editing and opportunities of nanomedicine. (n.d.). PMC. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC8316527/ Diabetes Prevention | ADA. (n.d.). Diabetes.org. Retrieved March 2\, 2026\, from https://diabetes.org/about-diabetes/diabetes-prevention Genetics of Diabetes. (n.d.). American Diabetes Association. Retrieved March 2, 2026, from https://diabetes.org/about-diabetes/genetics-diabetes Genome engineering and disease modeling via programmable nucleases for insulin gene therapy: Promises of CRISPR/Cas9 technology Eksi YE, Sanlioglu AD, Akkaya B, Ozturk BE, Sanlioglu S. Genome engineering and disease modeling via programmable nucleases for . (2021). Wignet. Retrieved February, 2025, from https://www.wjgnet.com/1948-0210/full/v13/i6/485.htm?appgw_azwaf_jsc=XU8ijqJujiB-gQuktGm7goqyBiz5k8CifpHmTOl6eCLiseAhIux08UV3V6zX-SStleeZzK8dwjNAKOx2BbQJwapX96t2B7RDukph5K1PMpmi4SoBC5hHsbzwlIq8_e35BkI9ocfyHe6JbIBWQQ_OKUz6CiBu8CeCP52kB3suqBzkDODQm7F9LmqTyol Glycemic Management in Adults With Type 1 Diabetes. (2018). Retrieved March 2, 2025, from https://guidelines.diabetes.ca/cpg/chapter12#bib0010 https://pmc.ncbi.nlm.nih.gov/articles/PMC6501476/. (2019, May 3). PubMedCentral. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC6501476/ Non Viral Vectors in Gene Therapy- An Overview. (2015, January 1). PMC. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC4347098/ Off-target effects in CRISPR/Cas9 gene editing. (n.d.). PMC. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC10034092/ Patient-Specific In Vivo Gene Editing to Treat a Rare Genetic Disease. (2025). Retrieved 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC12713542/ Srinivasan, M. (2021, December 19). Gene Therapy - Can it Cure Type 1 Diabetes? PMC. Retrieved March 2, 2026, from https://pmc.ncbi.nlm.nih.gov/articles/PMC8723777/ Type 1 diabetes - Diagnosis and treatment. (2024, March 27). Mayo Clinic. Retrieved March 2, 2026, from https://www.mayoclinic.org/diseases-conditions/type-1-diabetes/diagnosis-treatment/drc-20353017 What is Gene Therapy? (2025, February 14). YouTube. Retrieved March 2, 2026, from https://youtu.be/WmJAyyUqaZU

I would like to appreciate my mentors, Dr. Soares, Ms. Rachel, and Sophia Vig-Benko for supporting me through the countless hours of researching, editing and presenting. In addition, I appreciate the Renert science department for supporting all of the science fair competitors. Finally, I would like to thank my family for allowing me to spend time alone researching, and providing quiet spaces to allow me to focus. If it weren’t for all the support, I truly would not be where I am today, and I am extremely grateful.

Some content in this paper was clarified or organized with the assistance of AI tools, including ChatGPT (OpenAI) and Gemini (Google).