1. INTRODUCTION:

Gene therapy has become a groundbreaking method for treating both inherited and acquired diseases by allowing the direct modification of a patient’s genetic material. This therapeutic strategy is centred around the correction, replacement, or suppression of malfunctioning genes responsible for pathological conditions. Traditional gene therapy tools often relied on random gene insertion or non-specific modifications, leading to challenges in precision and safety. The introduction of genome editing technologies, particularly CRISPR-Cas systems, has transformed this field by providing a straightforward and highly precise approach to targeted genetic modification¹.

Initially identified as part of the bacterial adaptive immune system against viral infections, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has been adapted for genome editing in mammalian cells. This technique allows scientists and medical professionals to accurately modify specific genomic sequences by creating targeted double-stranded breaks, which are then repaired by the cell's natural DNA repair processes to achieve gene alterations².

A key advantage of the CRISPR-Cas9 system is in its programmability and in its flexibility, because it can enable genetic modifications within a wide variety of organisms and cell types. CRISPR is faster for design as well as supporting a wider array of targets, also it is simpler for use. It offers benefits beyond prior gene-editing methods such as zinc finger nucleases (ZFNs) and TALENs. Its ability for easy tailoring with guide RNAs has made it a leading choice for scientists researching and clinicians using it. Although CRISPR-based therapies hold great promise, their clinical success largely depends on the creation of effective, targeted, and safe delivery systems. It is essential that the Cas9 protein and guide RNA (gRNA) are delivered accurately to the intended tissues and cells, while avoiding degradation, immune responses, and off-target activity. Various delivery approaches have been investigated, including viral vectors, lipid nanoparticles (LNPs), polymer-based carriers, and physical techniques like electroporation and microinjection. Each method has its own unique benefits and drawbacks, which vary based on the disease type and target tissue³.

CRISPR-Cas technology enables precise genome editing with vast therapeutic potential, but its success depends on safe, efficient, and targeted delivery. Advances in viral, non-viral, and nanotechnology-based carriers are addressing these challenges. Clinically, CRISPR is being applied to treat genetic disorders, cancers, and infectious diseases, with emerging tools like base and prime editing expanding its scope. Optimized delivery strategies are key to translating these innovations into effective, targeted, and potentially curative gene therapies⁴.

2. Mechanism of CRISPR-Cas Gene Editing:

The CRISPR-Cas gene editing system is based on a bacterial immune mechanism used to defend against phage infections. In nature, bacterial cells incorporate snippets of invading viral DNA into their genomes, which are later transcribed into short RNA sequences that guide a Cas (CRISPR-associated) nuclease to recognize and cut the corresponding foreign DNA during future invasions.

In its therapeutic form, the CRISPR-Cas9 system is composed of two primary components:

1. Guide RNA (gRNA): A synthetically engineered RNA molecule that matches the target DNA sequence, directing the Cas protein to the exact genomic site by forming complementary base pairs.

2. Cas9 Nuclease: An enzyme obtained from Streptococcus pyogenes that functions to cut DNA. Once paired with the guide RNA, it creates a double-stranded break (DSB) at the exact DNA location targeted by the guide⁵.

Once the DSB is generated, the host cell's endogenous DNA repair mechanisms are activated. There are two primary repair pathways:

· Non-Homologous End Joining (NHEJ): This is the predominant repair mechanism in most mammalian cells. It rejoins the broken DNA ends without a template. Although efficient, NHEJ is error-prone and often introduces small insertions or deletions (indels), leading to gene disruption. This pathway is commonly used for gene knockout experiments.

· Homology-Directed Repair (HDR): This mechanism uses a homologous DNA template either endogenous or supplied exogenously—to precisely repair the break. HDR is employed for gene correction, replacement, or insertion. However, HDR is less efficient and limited to actively dividing cells.

The versatility of CRISPR allows for various genetic interventions, such as:

· Gene knockout: Useful for inactivating mutated genes that produce toxic proteins (e.g., Huntington's disease).

· Gene correction: For diseases caused by point mutations (e.g., sickle cell disease).

· Gene insertion or replacement: For inserting functional gene copies (e.g., hemophilia).

In addition to the classical CRISPR-Cas9 system, newer variations have been developed:

· CRISPR-Cas12 and Cas13 systems: These target single-stranded DNA or RNA, respectively, expanding the scope to include viral infections and RNA-level modulation.

· Base editors: These are specialized tools that enable the precise alteration of individual DNA bases (e.g., changing C to T or A to G) without causing double-stranded breaks in the DNA.

· Prime editing: An advanced method that allows accurate insertion, deletion, or substitution of DNA sequences, with reduced off-target activity and without requiring a donor DNA template.

Specificity and Targeting:

For accurate gene editing, the guide RNA (gRNA) must perfectly match the target DNA sequence positioned adjacent to a PAM (Protospacer Adjacent Motif), typically “NGG” for SpCas9. Even minor mismatches can lead to off-target effects, posing serious safety risks in clinical settings. To mitigate this issue, advanced gRNA design tools and enhanced Cas9 variants like SpCas9-HF1 and eSpCas9 have been developed to minimize unintended genetic modifications⁶.

Delivery Challenges and Packaging:

Efficient delivery of the CRISPR-Cas components to human cells is crucial for therapeutic outcomes. These components can be delivered in different forms:

· Plasmid DNA (encoding both gRNA and Cas9): Long-lasting but may cause insertional mutagenesis.

· mRNA (encoding Cas9) and synthetic gRNA: Fast expression with reduced immunogenicity.

· Ribonucleoprotein complexes (RNPs): Direct delivery of preassembled Cas9-gRNA complexes allows transient activity and reduces off-target effects.

Each form requires compatible delivery carriers such as adeno-associated viruses (AAVs), lipid nanoparticles, or exosome-based systems to navigate cellular and systemic barriers.

3. Delivery Strategies for CRISPR Components

One of the key challenges in gene therapy is the effective delivery of CRISPR components into cells. These components can be introduced in the form of plasmid DNA, mRNA, or protein complexes. The primary methods used for delivery include:

· Viral Vectors: Adeno-associated viruses (AAV), lentiviruses, and adenoviruses are frequently utilized because of their strong ability to deliver genetic material into cells. Nevertheless, issues like immune system reactions, restricted cargo capacity, and the potential for unintended integration into the genome pose limitations to their use.

· Non-Viral Methods: Lipid nanoparticles (LNPs), polymers, gold nanoparticles, and exosomes offer safer, customizable delivery mechanisms. LNPs have shown promise, particularly in delivering mRNA-based Cas9 systems with minimal toxicity.

· Physical Methods: Electroporation, microinjection, and hydrodynamic injection are effective in controlled lab settings but are less feasible for in vivo applications due to safety and scalability concerns.

· Cell-specific Targeting: Surface modifications and ligand attachment on nanoparticles can improve delivery to specific tissues or cell types, enhancing therapeutic precision.

· Ribonucleoprotein (RNP) Complexes: Delivering pre-assembled Cas9 protein with guide RNA offers rapid activity and transient expression, reducing off-target effects and immune responses compared to plasmid or mRNA formats.

· Virus-Like Particles (VLPs): These mimic viral entry mechanisms without containing viral genetic material, enabling efficient intracellular delivery with lower immunogenicity and no genomic integration.

· Cell-Penetrating Peptides (CPPs): Short peptides facilitate direct transport of CRISPR components across cell membranes, offering low toxicity and versatility for both in vitro and in vivo delivery.

· Hydrogel-Based Systems: Biocompatible hydrogels can encapsulate CRISPR components for sustained and localized release, suitable for tissue-specific applications like wound healing or tumour targeting.

· DNA Nanostructures: Engineered DNA origami frameworks serve as carriers that protect and direct CRISPR cargo precisely into target cells, enhancing stability and delivery efficiency.

4. Advantages of CRISPR-Based Therapeutics:

CRISPR offers numerous benefits for gene therapy:

· Precision and Efficiency: It allows for targeted gene modification with minimal off-target effects when properly designed.

· Multiplex Editing: Multiple genes can be edited simultaneously, enabling complex trait corrections.

· Versatility: Applicable to various cell types and disease models including somatic and germline cells.

· Reduced Cost: Compared to earlier genome editing tools like TALENs and ZFNs, CRISPR is more accessible and easier to produce.

5. Limitations and Challenges:

Despite its transformative potential, CRISPR technology presents several limitations:

· Off-Target Effects: Unintended mutations in the genome can cause oncogenic risks or disrupt essential genes.

· Immune Responses: Cas proteins, being foreign, may trigger immune responses in the host.

· Ethical Concerns: Germline editing raises moral and societal issues, especially in human embryos.

· Regulatory Hurdles: Variability in global regulations complicates clinical translation.

· Delivery Barriers: Safe and efficient delivery to specific tissues remains a bottleneck.

6. Real-Life Examples and Clinical Trials of CRISPR-Based Therapeutics:

The successful transition of CRISPR-Cas technology from bench to bedside has been marked by several pioneering clinical trials and real-world applications. Below are key examples demonstrating the current clinical impact and translational potential of CRISPR-based therapies.

6.1 Sickle Cell Disease (SCD):

A significant milestone in CRISPR-based gene therapy is its use in treating sickle cell disease, a hereditary blood disorder caused by a mutation in the β-globin gene. The investigational therapy exa-cel (formerly known as CTX001), developed by CRISPR Therapeutics and Vertex Pharmaceuticals, targets the patient’s own hematopoietic stem cells (HSCs). Through ex vivo CRISPR-Cas9 editing, the BCL11A gene responsible for suppressing fetal hemoglobin (HbF) production is inactivated. After reinfusion, the modified cells resume HbF production, effectively reducing or preventing sickling episodes. Clinical trials have demonstrated sustained therapeutic effects, with some patients remaining transfusion-free for more than 18 months⁷.

6.2 Cancer Immunotherapy:

CRISPR has opened a new frontier in oncology, particularly in immunotherapy. In China and the United States, clinical trials have explored editing T-cells to improve cancer-fighting capacity. A notable application includes the removal of PD-1 (programmed death-1) receptors using CRISPR-Cas9 to prevent immune exhaustion and enhance T-cell-mediated cytotoxicity against tumours. This strategy has been tested in patients with non-small cell lung cancer (NSCLC), multiple myeloma, and sarcomas. The preliminary results indicate feasibility and tolerability, although the long-term efficacy and safety remain under evaluation⁸.

6.3 Leber Congenital Amaurosis (LCA10):

EDIT-101, developed by Editas Medicine, represents the first in vivo CRISPR therapy to reach clinical trials for inherited retinal disorders. The treatment targets CEP290 gene mutations, a major cause of LCA10, an early-onset form of blindness. Using an AAV (Adeno-Associated Virus) vector, the CRISPR components are directly injected into the subretinal space. Preclinical results showed significant restoration of photoreceptor function, and early human trials have demonstrated encouraging trends in visual improvement.

6.4 Beta-Thalassemia:

Similar to SCD, β-thalassemia is addressed using CRISPR-Cas9 to reactivate fetal haemoglobin production. In trials analogous to those of exa-cel, HSCs are harvested, edited ex vivo, and re-infused after myeloablation. The edited cells express increased HbF, compensating for the defective β-globin. Patients in Phase I/II trials have shown a marked reduction in transfusion dependence and improved haemoglobin levels, showcasing the potential for a functional cure.

6.5 CRISPR in COVID-19 Diagnostics:

Beyond gene editing, CRISPR has significantly contributed to the progress of molecular diagnostics. Platforms like SHERLOCK (Specific High-sensitivity Enzymatic Reporter Unlocking) and DETECTR use CRISPR-associated enzymes such as Cas13 or Cas12 to detect viral RNA, including that of SARS-CoV-2. These technologies enable rapid, accurate, and affordable testing at the point of care, making them invaluable tools for managing and containing pandemics⁹.

7. Ethical and Regulatory Perspectives:

The rise of CRISPR-based gene editing has prompted intense ethical discussions, particularly regarding modifications to the human genome. Editing somatic cells those that are non-reproductive has largely been accepted by the scientific community and regulators when conducted under strict oversight. In contrast, germline editing, which involves making heritable genetic changes, remains highly controversial. Key concerns include the risk of unforeseen long-term effects, such as mosaicism and off-target mutations, as well as ethical dilemmas about consent from future generations.

Another layer of complexity stems from the possibility of “designer babies,” where editing might be pursued for enhancement rather than therapeutic intent. Global voices, including scientists, bioethicists, and policymakers, have stressed the need for international regulatory consensus. High-profile controversies such as the 2018 case in China where gene-edited embryos were implanted have fuelled global calls for moratoriums and stricter oversight.

Regulatory frameworks are still evolving. In the United States, the FDA oversees gene editing products, ensuring compliance with safety and ethical standards, while the NIH provides additional review through its Recombinant DNA Advisory Committee. In Europe, the European Medicines Agency (EMA) and national ethics councils collaborate to assess both scientific and ethical dimensions of gene therapy trials. Key focus areas include patient consent, transparency in clinical outcomes, long-term monitoring, and equitable access to novel treatments¹⁰.

Additionally, bioethics boards and institutional review committees are tasked with ensuring that informed consent protocols fully capture the experimental nature of such therapies. Discussions are ongoing about developing international registries, enhancing patient advocacy participation, and formulating global ethical guidelines to govern the responsible deployment of CRISPR in both somatic and germline contexts¹¹.

8. Future Directions:

CRISPR-based gene editing holds immense potential to revolutionize biomedicine, with ongoing research aimed at improving its safety, accuracy, and therapeutic scope. Significant advancements include the development of next-generation tools like base editors and prime editors, which allow precise genetic modifications without creating double-stranded DNA breaks. Base editors facilitate specific A-T to G-C or C-G to T-A conversions, while prime editors can accurately insert, delete, or replace DNA sequences with fewer off-target effects. These innovations are especially promising for correcting point mutations that cause various monogenic disorders.

Nevertheless, precision in gene editing is not enough effective delivery systems remain a significant challenge. While traditional viral vectors like adeno-associated viruses (AAVs) offer high efficiency, they can provoke immune reactions and have limited capacity for genetic cargo. As a result, researchers are actively exploring non-viral delivery methods, including lipid nanoparticles (similar to those used in mRNA vaccines), gold nanoparticles (like CRISPR-Gold), exosomes, and polymer-based nanocarriers. These alternatives show promise for delivering CRISPR components to difficult-to-access tissues, such as the brain or muscles, with lower risk of immune response. Furthermore, spatiotemporal control methods, such as light-activated or chemically triggered CRISPR systems, are being developed to enable gene editing at precise times and locations within the body¹².

The incorporation of artificial intelligence (AI) and machine learning (ML) is rapidly enhancing all facets of CRISPR research. AI-driven platforms are being used to predict off-target effects, optimize guide RNA (gRNA) sequences, model protein–DNA interactions, and simulate repair outcomes post-editing. These computational approaches significantly reduce the trial-and-error phase of gene-editing experiments and support the development of personalized medicine strategies.

Additionally, CRISPR is being combined with induced pluripotent stem cells (iPSCs) to develop personalized disease models tailored to individual patients. These platforms enable scientists to study disease mechanisms in a genetically controlled environment and test CRISPR-based corrections ex vivo before clinical translation. This synergy between gene editing and stem cell technology is especially promising for complex diseases like Alzheimer’s, Parkinson’s, and various cardiovascular disorders.

Further ahead, synthetic biology is contributing to the design of programmable gene circuits where CRISPR systems can be toggled or modulated based on cellular signals. These “smart” therapies could autonomously respond to disease biomarkers and apply edits only when needed, reducing systemic exposure and side effects.

Taken together, these advances are pushing CRISPR toward clinical maturity. With collaborative efforts across disciplines biotechnology, nanotechnology, AI, and ethics CRISPR has the potential to reshape not only how we treat disease but how we understand and engineer life at the molecular level¹³.

9. CONCLUSION:

CRISPR-Cas systems mark a transformative advancement in therapeutic science, providing the ability to address genetic disorders at their source. This single programmable platform allows scientists to modify, remove, or repair defective genes, opening the door to a new era of curative treatments. The positive outcomes from initial clinical trials targeting conditions such as sickle cell disease, β-thalassemia, and retinal dystrophies highlight the tangible benefits of this groundbreaking technology.

However, challenges persist. Efficient and safe delivery remains a bottleneck, off-target effects still require mitigation, and public trust must be earned through ethical governance. The balance between innovation and regulation is crucial. If navigated responsibly, CRISPR has the potential not only to treat monogenic diseases but also to reshape the management of complex conditions such as cancer, viral infections, and neurodegenerative disorders.

In summary, CRISPR-based drug delivery and therapeutics mark a profound leap toward curative and precision-driven healthcare. With ongoing technological refinement, ethical oversight, and interdisciplinary collaboration, CRISPR will likely become a cornerstone of modern medicine in the decades to come.

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Received on 11.08.2025 Revised on 13.09.2025

Accepted on 09.10.2025 Published on 18.10.2025

Available online from November 03, 2025

Res. J. Pharma. Dosage Forms and Tech.2025; 17(4):273-278.

DOI: 10.52711/0975-4377.2025.00038

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