INTRODUCTION:

The CRISPR-CRISPR-associated protein 9 (Cas9) system was initially created by bacteria as a defence mechanism against phage infection and plasmid transfer in nature. However, it has since been repurposed as a powerful RNA-guided DNA targeting platform for genome editing, transcriptional perturbation, epigenetic modulation, and genome imaging. It also enables the inhibition of activated oncogenes or the activation of deactivated cancer suppressor genes, the repair of illness-causing mutations, and the clarification of gene function implicated in the onset and course of disease¹.

Gene therapy now has a fresh chance to transcend past stigmas and restrictions thanks to the discovery and development of the CRISPR/Cas9 system, establishing it as an effective therapeutic approach. Nonetheless, there are a number of technical and moral issues with the use of CRISPR technology in patient care that must be properly considered².

Because CRISPR/Cas can accurately target and edit individual genes within a genome, it has emerged as a formidable tool for genetic manipulation. After being translated into a lengthy RNA molecule, the CRISPR array is broken down into shorter CRISPR RNAs (crRNAs), each of which has a distinct spacer sequence that is obtained from earlier viral infections. A ribonucleoprotein (RNP) complex is formed when these crRNAs are joined with a tiny trans- activating CRISPR RNA (crRNA) complex. With the help of the spacer sequence in the crRNA, this RNP complex locates and binds to complementary regions in the target DNA. As soon as a match is discovered, the Cas9 protein breaks the DNA double-stranded break (DSB) by functioning as a nuclease.³

Gene therapy and hematopoietic stem cells:

Because of their great potential for lifespan and ability to self-renovate, haematopoietic stem cells have emerged as promising candidates for gene transfer. The synthesis of gene transfer vectors for the generation of induced pluripotent stem cells (IPS), which allows for the differentiation of the IPS and the provision of an extra phenotype from this differentiated derived cell, is one example of this coupling of gene therapy and stem cells. In addition, it need to be safe for the experts handling it as well as the surroundings and the patient. Lastly, the vector needs to be able to express the gene generally for the duration of the patient's life⁴.

CRISPR-Cas9:

A unique pattern in the 1980s was found in a section of the Escherichia coli genome where a highly variable sequence was intercalated by a repeating sequence that had no known purpose. This led to the discovery of the then-unknown CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (Associated Proteins), which have become one of the main biotechnological tools for gene editing since 2012⁵.

The American biologist Jennifer Doudna and the French microbiologist Emmanuelle Charpentier were given the Nobel Prize in Chemistry in October 2020 for "developing a new approach to genome editing." The CRISPR technology was discovered as a result of an unforeseen incident, similar to many other notable breakthroughs. When researching the E. coli gene in 1987, Nakata et al. discovered an odd sequence in the 3′ end structural domain of the gene. The sequence was made up of five extremely similar sequences, each with 29 nucleotides and 32 nucleotides between them⁶.

The CRIPSR technology was created based on this natural mechanism and allows editing of target specific DNA sequences in any organism's genome using just three molecules: nuclease, specifically caspase 9, which cleaves double-stranded DNA; the target DNA; and an RNA guide, which directs the complex to the target⁷.

Strategies Of Crispr/Cas9‐Based On Gene Therapy:

CRISPR/Cas9-based gene therapy can employ any CRISPR Cas technique, which can be utilised to eliminate, swap out, or fix unwanted genes that cause hereditary illnesses. CRISPR/Cas9 technology has evolved in less than ten years, despite the fact that it was acknowledged as a genome editing tool in 2012. Numerous techniques have been created and used in a range of basic and practical research projects. Among these, prime editing, base editing, and gene knocking /out have demonstrated extraordinary promise in gene therapy⁸.

Figure 1. Major strategies for CRISPR/Cas9 gene therapy.

Mechanistic Overview of CRISPR/Cas9-Mediated Genome Editing:

A straightforward two-component method for efficient targeted gene editing is called CRISPR/Cas9. HNH cleaves the complementary DNA strand to the spacer sequence, while the CRISPR/Cas9 complex gets directed to the desired chromosomal region by this sgRNA. The editing system then uses homology-directed repair (HDR) or non-homologous end-joining (NHEJ) as its two natural DNA repair mechanisms. NHEJ, which involves the random insertion and deletion of base pairs, or indels, at the cut site, happens far more frequently in the majority of cell type.

The other approach is the error-free HDR pathway, which is particularly desirable to use for therapeutic purposes. This mechanism can be used in experiments by facilitating the desired modification into the genome with an external donor template that has the CRISPR/Cas9 machinery⁹.

Double-stranded breaks (DSBs) are caused by the Cas-9 nuclease at a location three base pairs upstream of PAM¹⁰.

The target DNA is broken on both strands by the Cas9 protein using its RuvC and (HNH) domains. For Cas9 to break DNA, the PAM sequence is primarily required. 20 bases where the selectivity for binding in sgRNA is found. DNA double strand breaks (DSBs) can be repaired in two ways: either by gene replacement or knock-in, or by homology-directed repair (HDR), which needs a template and results in non-homologous end-joining (NHEJ), a loose but permanent knockout of a gene¹¹.

When Cas9 and sgRNA form a complex, the corresponding gene is targeted, and DSBs are produced close to the PAM region. One of the two pathways used for DNA damage repair is the NHEJ pathway or HDR. Error-prone repair is produced by random insertions and deletions (indels) that are inserted at the cut side and ligated in the NHEJ pathway. Error-free repair is achieved through the homologous chromosomal DNA acting as a template for the damaged DNA during the HDR pathway.

Biological Intervention of CRISPR/Cas9 in Clinical Trials:

Cancer Immunotherapy:

In order to employ CRISPR/Cas9 to modify autologous T cells for cancer immunotherapy against a number of diseases with relapsed tumours and no other viable therapeutic choices, the first ^CRISPR Phase 1 clinical trial in the US was launched in 2018. These comprise myxoid/round cell liposarcoma, melanoma, synovial sarcoma, and multiple myeloma. The US Food and Drug Administration (FDA) gave this trial approval after carefully weighing the risks and benefits of this initial clinical use of CRISPR gene therapy.

CRISPR-mediated cancer immunotherapy has been the subject of numerous trials and is presently the most widely used CRISPR gene therapy technique. Although a pre-clinical and clinical experiment employing this approach with alternative instruments has previously been carried out, this was the first time that CRISPR/Cas9 was utilised to create the genetically altered T cells¹².

Gene Disruption:

Vertex Pharmaceuticals and CRISPR Therapeutics performed the first clinical trial in the US for patients with sickle-cell anaemia (SCD) and later β-thalassemia, utilising CRISPR to catalyse gene disruption for therapeutic benefit. The treatment known as CTX001 raises the amount of foetal haemoglobin (HBF), which can occupy one or two of the four haemoglobin binding pockets on erythrocytes. This helps treat severe β-haemoglobin illnesses like β- thalassemia and sickle cell disease (SCD) clinically¹³.

In vivo CRISPR Gene Therapy:

A promising advancement in CRISPR gene therapy has been initiated through a clinical trial that employs in vivo delivery of CRISPR/Cas9 in patients for the first time. Some organs, including the eye, are accessible, but in vivo editing has been mainly restricted by poor access to the target tissue. There is no known cure for the crippling monogenic condition known as Leber congenital amaurosis (LCA), which is brought on by a bi-allelic loss-of-function mutation in the CEP290 gene and causes juvenile blindness. The medication, which is called EDIT-101, particularly targets individuals with the intronic IVS26 mutation in the retina, causing incorrect splicing that results in a non-functional protein¹⁴.

CRISPR Editing in Human Embryos and Ethical Considerations:

Human germline editing for therapeutic purposes is still quite controversial, even though somatic editing for CRISPR therapy has been authorised after considerable thought. Any possible risk with somatic edition would be contained inside the patient once they gave their informed agreement to get the therapy. Not only does embryonic editing rob later-born humans of their autonomy in making decisions, but it also allows unintended and irreversible adverse effects to be passed down through generations¹⁵.

Potential for CRISPR therapeutics during COVID-19 pandemic:

The quick development of CRISPR technology might be helpful in these quickly changing times. A worldwide pandemic has resulted from the recent breakout of a novel strain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to provide patients with COVID-19 with prompt and effective testing and treatment choices, there is an urgent need to respond to these dire circumstances. The testing techniques that are currently available take a long time and have poor sensitivity and accuracy¹⁶.

CRISPR/CAS9 Has Great Potentials On Gene Therapy:

CRISPR Cas system is a robust and powerful tool not only for altering a gene for its expression and function, but also for screening, monitoring, and regulating those genes. Since it was considered as a genome editing tool in 2012, CRISPR Cas system has been widely used in studying gene function in human and/or human‐related traits, particularly on hu man development and genetic disease‐related aspect The past 8 years of CRISPR/Cas‐related studies have shown its huge potentials on gene therapy, and CRISPR/Cas9 genome editing can be employed in treating any human diseases associated with genetic mutation or genetic element change (Figure 5)⁸.

Fiture 5. Huge potentials of CRISPR Cas genome editing in treating human genetic diseases.

First, CRISPR/Cas9 is becoming a powerful tool for high throughput screening for target genes for cancer therapy, in which gRNA libraries were used to target many potential genes for loss of function for studying the cell responses, such as accelerated metastasis, influencing immune response and drug resistance.¹⁷.

CRISPR/Cas potentially treats human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS):

HIV attacks human immune cells and finally leads to the AIDS. Recently developed CRISPR/Cas9 provides new hopes for against HIV and treating human AIDS. As HIV gene is regulated by the long terminal repeat (LTR) promotor when HIV genome is inserted into the host genome, LTR is becoming a target region for HIV treatment¹⁸.

CRISPR/Cas potentially treats cardiovascular diseases (CVDs):

CVDs have attracted more and more attentions form scientific and industrial communities. Because many factors associating with CVDs are related to genetic controls, CRISPR/Cas9 genome editing is also given promising hope for treating CVDs. It is well‐known that high blood lipid level is linked to CVDs; reduced blood lipid levels significantly reduced the risk of, CVD¹⁹.

CRISPR/Cas potentially treats ocular diseases:

ocular diseases have been attracting more and more attention from scientific and bio medical communities as well as industries. Because many eye dis orders are associated with genetic elements, such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA2), X‐linked retinoschisis, and choroideremia, CRISPR/Cas9‐based gene therapy also has huge potential for treating genetics‐related ocular diseases²⁰.

CRISPR/Cas Potentially Treats Viral Infectious Diseases:

The prevention of viral infection or replication is included in antiviral genome editing. One of the most promising and emerging gene therapy techniques is programmable nuclease-mediated antiviral treatment. In order to inhibit viral infection, Cas9 nucleases can target viral genes or host genes that encode critical receptors, such as HIV-1, Epstein–Barr Virus, Hepatitis B Virus, Herpes Simplex Virus, Human Papillomavirus, etc²¹.

CRISPR/Cas Potentially Treats Genetic Diseases:

The CRISPR Cas system has been widely used in the correction of human genetic diseases including Duchenne muscular dystrophy (DMD), α-1 antitrypsin deficiency (AATD), hemophilia, hematopoietic diseases, and hearing loss. The genetic corrections are carried out by CRISPR–Cas9-based hematopoietic stem and progenitor cells (HSPCs), recovering pathogenic mutation in induced pluripotent stem cells (iPSCs) in normal hemoglobin, etc²².

CRISPR–Cas9: Ifs and Buts:

A major limitation is the production of off-target effects in host cells, especially in mice embryos and adult human cells. The sgRNA along with Cas9, despite widely used in genome editing, are limited by off-target effects and chromosomal translocation due to off-target cleavage. Scientists have used plasmids, viruses, and ribonucleoproteins for delivery purposes, but the process also suffers from limitations²³.

Due to its universality, CRISPR will probability also be applied in animals to increase muscle mass, to reduce diseases, to improve vitality, etc. However, much more dangerous is the application of CRISPR to eradicate diseases by eradicating disease vectors and invasive species. One of the examples is Aedes aegypti, a mosquito that transmits dengue fever. Researchers are developing genetically edited male-sterile mosquitos to prevent reproduction, with an aim to reduce the spread of disease²⁴.

One of the most discussed topics associated with CRISPR is human genome editing. There are three main discussed problems:

1. Risk and uncertainty of the technology and its application.

2. The human germline interference and responsibility towards future generations, and

3. The legitimization of human genome editing measures with regard to concepts of therapy and enhancement²⁵.

Future Perspective:

CRISPR/Cas9 as a microbial intrinsic immunity framework has been developed as a robust gene-editing technology. Due to its precision and efficiency, CRISPR/Cas9 techniques can provide an incredible chance to treat a few gene-related diseases by deletion, insertion, regulation, and blocking different genes. Cas9-mediated gene therapy has been utilized to treat different non-cancerous maladies in some studies, CAR-T cells have been generated by the Cas9 system and reached successfully to clinical trial phases. In some cases, a few issues keep unsolved like off-target effects, transfer challenges, PAM confinement, and immunogenicity²⁶_.

Within the last few years we have witnessed stunning progress in the development of various CRISPR-based technologies. The therapeutic applications of the CRISPR technologies are particularly exciting²⁷.

CONCLUSION:

CRISPR/Cas9 technology has been used more and more Widely for genome editing. Its efficacy, specificity, easy to Use, cost-effectiveness and versatility, will boost this technique on more fronts. In this review, we examined various Aspects of sgRNA design tools, including activity prediction Models, and off-target detection algorithms. Almost all of These models or algorithms depend on large-scale experimental datasets and systematic analysis. Gene therapy and CRISPR technology are at the forefront of a genetic revolution, offering hope for curing previously untreatable diseases. As research progresses, the potential for these technologies to transform medicine and improve human health is immense. However, the careful consideration of the ethical and safety implication is essential to ensure that these powerful tools are used responsibly.

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Received on 17.04.2025 Revised on 16.06.2025

Accepted on 26.07.2025 Published on 04.10.2025

Available online from October 10, 2025

Asian J. Res. Pharm. Sci. 2025; 15(4):399-404.

DOI: 10.52711/2231-5659.2025.00059

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

CRISPR Cas 9 Technology: Future of Gene Editing