Revolutionizing healthcare: the role of CRISPR-Cas systems in precision medicine

Introduction

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and represents a groundbreaking advancement in genetic engineering. Initially identified in E. coli in 1987, these unique DNA sequences were characterized by their distinctive pattern of short, repetitive repeats interspersed with spacer sequences [1]. Although their function has remained elusive, subsequent research has revealed their role in the bacterial adaptive immune system, which provides protection against viral invaders.

The transformative potential of CRISPR in modern medicine is profound. The unparalleled precision and efficiency of genome editing have opened new avenues for correcting genetic mutations responsible for various diseases [2]. Beyond therapeutic applications, CRISPR-based diagnostic tools have been developed, offering rapid and accurate detection of pathogens and genetic anomalies. The adaptability of CRISPR systems extends to diverse fields, including agriculture and environmental science, highlights their versatility [3].

This review comprehensively examines the transformative potential of CRISPR in disease diagnosis and treatment. We will explore the origins and mechanisms of CRISPR technology, delve into its applications in disease diagnostics and therapeutics, discuss emerging innovations, and address the ethical and societal considerations accompanying its rapid advancement. By clarifying these facets, we highlight the pivotal role that CRISPR plays in shaping the future of precision medicine.

Methodology

This literature review adhered to a systematic approach in line with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines to ensure a transparent and reproducible study selection process. The study identified, analyzed, and synthesized existing research on CRISPR-Cas systems in precision medicine. The following steps were taken:

Search strategy

A comprehensive search was conducted in four prominent scientific databases: PubMed, Scopus, Google Scholar, and Web of Science. The search strategy was designed to retrieve articles related to CRISPR-based diagnostics, therapeutic applications, and technological advancements. The search included the following keywords and Boolean operators:

• "CRISPR-based diagnostics" AND "precision medicine"
• "Gene editing" AND "CRISPR-Cas technologies"
• "CRISPR-based innovation" OR "CRISPR therapeutic applications"
• "Emerging trends in CRISPR technology"

To minimize publication bias, the gray literature and reference lists of the included articles were also screened.

Inclusion and exclusion criteria

The inclusion criteria ensured a focus on high-quality and relevant studies:

• Articles published in peer-reviewed journals.
• Publications in English from 2015 to 2025.
• Studies specifically addressing CRISPR-Cas systems in diagnostic or therapeutic applications.
• Clinical or translational advancements in precision medicine.

The exclusion criteria filtered out studies:

• Not directly related to CRISPR systems.
• Published in non-English languages.
• Lack of sufficient methodological details or primary research focus.

Study selection process

A total of 139 records were retrieved through the initial database search. Duplicate entries were identified and removed using EndNote. The remaining 83 unique articles underwent a two-phase screening:

1. Title and Abstract Review: Articles were assessed for relevance based on predefined inclusion criteria. This process resulted in 46 eligible studies.
2. Full-Text Screening: Each study was evaluated in detail to confirm its alignment with the review objectives. This phase also included a critical appraisal of the study quality and methodology.

Two independent reviewers screened the studies based on predefined inclusion and exclusion criteria. Discrepancies were resolved through discussion, and a third reviewer was consulted in cases of disagreement.

Quality assessment

Each study was assessed using the CASP framework with a scoring system to evaluate methodological rigor, sample size, and reproducibility. Studies with scores below 50% were excluded. Key elements included:

• Validity of the research design.
• Reproducibility of results.
• Relevance to CRISPR applications in precision medicine.

Publication bias was assessed using funnel plot asymmetry and Egger’s test to ensure a comprehensive evaluation of bias in the selected literature.

Data extraction and synthesis

Data from the included studies were systematically extracted and synthesized. Key variables included:

• Study objectives, design, and methods.
• CRISPR system type (e.g., Cas9, Cas12, Cas13).
• Applications of the proposed technologies to diagnostics, therapeutics, and emerging technologies.
• Ethical and regulatory considerations.

Data extraction was conducted independently by two reviewers, and the accuracy was verified by cross-checking. The key variables were tabulated, and inconsistencies were resolved through discussion.

The findings were categorized into thematic areas for analysis, including diagnostic tools, therapeutic advancements, and emerging innovations.

CRISPR as a diagnostic tool

CRISPR-based diagnostic tools have emerged as revolutionary platforms for molecular diagnostics, offering rapid, sensitive, and specific detection of nucleic acids. Among these, SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) stand out for their innovative applications. SHERLOCK utilizes the collateral cleavage activity of the Cas13 enzyme upon binding to its target RNA sequence, enabling the detection of specific RNA sequences with high sensitivity [4]. Similarly, DETECTR employs Cas12, which, upon recognition of a target DNA sequence, exhibits collateral cleavage activity, facilitating the detection of specific DNA sequences. These platforms have been adapted for various diagnostic purposes, including the detection of viral and bacterial pathogens and genetic mutations associated with diseases [5].

The key advantages of CRISPR-based diagnostics compared with conventional methods are noteworthy. Traditional diagnostic techniques, such as polymerase chain reaction (PCR), are sensitive; however, they often require complex instrumentation, longer processing times, and are susceptible to contamination, leading to false-positive results [6]. In contrast, CRISPR-based assays like SHERLOCK and DETECTR, offer rapid detection, often within an hour, with minimal equipment requirements. Their high specificity is attributed to the programable nature of the CRISPR system, which can be tailored to recognize unique genetic sequences, thereby reducing the likelihood of cross-reactivity and false positives [7]. In addition, these assays have demonstrated the capability to detect low levels of target nucleic acids, enhancing their sensitivity and making them suitable for early-stage disease detection.

In infectious disease diagnostics, CRISPR-based tools have shown significant promise. For instance, during the COVID-19 pandemic, CRISPR-based assays were developed for detecting SARS-CoV-2, which is the virus responsible for COVID-19. These assays used the DETECTR platform to identify the presence of viral RNA in patient samples, offering a rapid and accurate alternative to traditional PCR-based tests [8]. Beyond viral infections, CRISPR-based diagnostics have been applied to detect bacterial pathogens, such as Escherichia coli and Staphylococcus aureus, as well as fungal infections, demonstrating their versatility across a broad spectrum of infectious agents [9].

The real-world applications of CRISPR-based diagnostics have been instrumental in epidemic management. The rapid development and deployment of CRISPR-based tests during the COVID-19 pandemic exemplifies their potential for use in outbreak scenarios [10]. These tests provided a means for widespread screening, enabling the timely identification and isolation of infected individuals, thereby contributing to containment efforts. Furthermore, the adaptability of CRISPR-based diagnostics allows for quick reconfiguration to detect emerging pathogens, making them valuable tools for responding to future infectious disease threats [11].

In cancer diagnostics, CRISPR-based methods have been explored for the detection of genetic mutations and biomarkers associated with various malignancies. By designing CRISPR systems to target specific oncogenic mutations, researchers have developed assays capable of identifying cancer-related genetic alterations with high precision [12]. Additionally, CRISPR-based approaches have been applied to liquid biopsies, which involve the analysis of circulating tumor DNA (ctDNA) in bodily fluids like blood. These non-invasive tests can detect early cancer, monitor disease progression, and assess treatment response, thereby contributing to personalized cancer management strategies [13].

Despite these promising advantages, several challenges hinder the widespread clinical implementation of CRISPR-based diagnostics. Cost considerations remain a significant barrier because the development and production of these assays can be expensive, potentially limiting accessibility, especially in resource-limited settings [14]. Scalability is another concern, as the transition from laboratory-based assays to high-throughput clinical testing requires robust validation and standardization processes. Regulatory hurdles also pose challenges, as the approval and integration of new diagnostic technologies into clinical practice require rigorous evaluation to ensure safety, efficacy, and reliability [15].

CRISPR in therapeutics

CRISPR-Cas9 gene editing has emerged as a transformative approach for treating monogenic diseases—disorders caused by mutations in a single gene. Sickle cell disease (SCD) and cystic fibrosis (CF) are prominent examples of these conditions [16]. In SCD, a mutation in the β-globin gene leads to abnormal hemoglobin formation, causing red blood cells to assume a sickle shape, resulting in various complications. CRISPR-based therapies aim to correct this mutation or reactivate fetal hemoglobin production to mitigate disease symptoms [17]. Recent advancements have led to the approval of gene therapies using the CRISPR-Cas9 technology for SCD treatment, marking a significant milestone in the application of genome editing in clinical settings [18].

In the realm of cancer therapy, CRISPR has been instrumental in the engineering of T cells to enhance their efficacy against malignancies. Chimeric Antigen Receptor T-cells (CAR-T cells) are modified to express receptors that target specific cancer antigens [19]. CRISPR-Cas9 facilitates the precise editing of T-cell genomes, improving their persistence and reducing exhaustion, thereby enhancing their anti-tumor potential. This approach has shown promise in preclinical studies, suggesting that CRISPR-mediated modifications can optimize CAR-T cell therapies and improve clinical outcomes [20].

In addition to oncology, CRISPR is being explored as a therapeutic tool against infectious diseases. For chronic infections like Hepatitis B virus (HBV), CRISPR-Cas9 has been used to target and disrupt viral DNA within host cells [21]. Preclinical studies have demonstrated the feasibility of this approach, showing that CRISPR-mediated gene editing can reduce viral loads in models of chronic HBV infection. However, challenges such as efficient delivery and off-target effects need to be addressed before clinical application [22].

The use of CRISPR to treat polygenic and complex disorders, such as neurodegenerative diseases, is an area of active research. Neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases, involve multiple genetic and environmental factors [23]. CRISPR-Cas9 has demonstrated potential in preclinical models by targeting genes implicated in these disorders, offering a promising avenue for therapeutic intervention. Nonetheless, the complexity of these diseases poses significant challenges, including the need to target multiple genes and regulatory networks and ensure precise delivery to affected tissues [24].

Emerging innovations in CRISPR technology

Recent advancements in CRISPR technology have led to the development of next-generation gene-editing tools, notably base and prime editing tools, which offer significant advantages over the traditional CRISPR-Cas9 system. Base editing enables precise single-nucleotide conversions without the introduction of double-strand breaks (DSBs) [25]. For instance, cytosine base editors (CBEs) can convert a C•G base pair to T•A base pairs, whereas adenine base editors (ABEs) facilitate the conversion of A•T to G•C. This precision reduces the risk of unintended insertions and deletions (indels) associated with DSB repair mechanisms [26]. Prime editing further extends this capability by allowing not only base substitutions but also small insertions and deletions without creating DSBs. This method employs a fusion of catalytically impaired Cas9, reverse transcriptase, and prime editing guide RNA (pegRNA) to direct the desired edit, offering a versatile and efficient approach to genome modification [27].

The CRISPR-Cas system encompasses various Cas proteins, each with unique properties suitable for specialized applications. For example, Cas12 introduces staggered DNA fragments that are advantageous for certain types of genetic modifications [28]. Its smaller size compared with Cas9 facilitates more efficient delivery into cells. Conversely, Cas13 targets RNA instead of DNA, making it a valuable tool for applications requiring RNA manipulation, such as transcriptome engineering and antiviral strategies [29].

Efficient and safe delivery of CRISPR components into target cells remains a challenge in in vivo applications. Recent progress in delivery mechanisms has shown promise in overcoming these obstacles. Viral vectors, such as adeno-associated viruses (AAVs), have been widely used because of their high transduction efficiency and ability to infect a broad range of cell types [30]. However, their limited packaging capacity and potential immunogenicity have spurred the development of non-viral delivery methods. Lipid nanoparticles (LNPs) have emerged as a promising alternative with advantages such as lower immunogenicity, the capacity to carry larger genetic payloads, and the ability to deliver CRISPR components in the form of ribonucleoprotein complexes, which can reduce off-target effects [31]. Advancements in delivery systems are crucial for translation of CRISPR-based therapies into clinical settings.

Ethical and societal considerations

The advent of the CRISPR-Cas9 gene editing technology has ignited significant ethical and societal debates, particularly concerning its application in human germline editing. Altering the germline—the genetic material passed to future generations—raises profound ethical dilemmas [32]. One primary concern is the potential for "designer babies," where genetic modifications can be used to select desired traits, thereby worsening social inequalities and leading to unforeseen societal consequences. In addition, the possibility of unintended off-target effects increases the risk of introducing new genetic anomalies into the human gene pool [33].

The regulatory frameworks governing CRISPR applications vary significantly across countries, reflecting diverse ethical stances and policy approaches. Since 2014, approximately 40 countries, including 15 from Western Europe, have prohibited research on germline editing because of ethical and safety concerns. International efforts, such as the summit led by the United States, the UK, and China in 2015, aim to harmonize regulations and establish guidelines for responsible genome-editing practices [34, 35].

Equity in access to CRISPR-based therapies presents another critical challenge. The high costs associated with these treatments, exemplified by therapies priced at around $2 million per patient, raise concerns about affordability and accessibility, particularly in low- and middle-income countries [36]. This disparity underscores the need for strategies to ensure that gene editing advancements do not worsen existing health inequities but instead contribute to global health improvements [37].

Current challenges and future directions

The CRISPR-Cas9 gene-editing system has revolutionized biomedical research and holds immense potential for therapeutic applications. However, several technical challenges must be addressed to fully realize its clinical utility. One primary concern is the occurrence of off-target effects, where Cas9 nuclease introduces unintended modifications at genomic sites resembling the target sequence [38]. These off-target mutations can lead to unpredictable consequences, including disruption of essential genes or activation of oncogenes. Strategies to mitigate these effects include the development of high-fidelity Cas9 variants and the use of computational tools to design more specific guide RNAs [39].

Another significant hurdle is the immune response induced by the introduction of CRISPR components into the human body. Cas9 protein, derived from bacterial species, can be recognized as foreign by the human immune system, potentially leading to immune reactions that diminish the efficacy of the therapy or cause adverse effects. Research is ongoing to engineer Cas9 proteins with reduced immunogenicity and to explore transient delivery methods that minimize immune activation [40, 41].

The scaling up of CRISPR applications presents additional challenges, particularly in the context of manufacturing and delivering gene-editing components for widespread clinical use. Efficient delivery systems are crucial for ensuring that CRISPR components reach target cells in sufficient quantities without causing toxicity. Advancements in nanoparticle-based delivery methods and viral vectors are being explored to enhance delivery efficiency and scalability [12, 42].

The translation of CRISPR technology from laboratory research to real-world therapies and diagnostics involves overcoming several bottlenecks. Preclinical studies must thoroughly assess the safety and efficacy of CRISPR-based interventions, which requires the development of reliable animal models and comprehensive genomic analyses to detect off-target effects [43]. Regulatory frameworks for gene-editing therapies are still evolving, necessitating clear guidelines to ensure patient safety while fostering innovation. Additionally, large-scale manufacturing processes need to be established to produce clinical-grade CRISPR components under Good Manufacturing Practice (GMP) conditions [44].

Despite these promising advancements, several challenges remain, including the risk of off-target effects in CRISPR applications, ethical concerns, and limitations in large-scale clinical translation. Future studies should focus on addressing these gaps through enhanced specificity and regulatory frameworks.

The next decade will see a significant impact of CRISPR technology on personalized medicine and global health. In personalized medicine, CRISPR can be used to tailor treatments based on individual genetic makeup, allowing for the correction of specific mutations responsible for the disease [45]. For instance, ex vivo editing of patient-derived cells followed by autologous transplantation has been successful for treating certain blood disorders. In the realm of global health, CRISPR-based diagnostics offer rapid, sensitive, and cost-effective tools for detecting infectious diseases, which are particularly valuable in resource-limited settings. Furthermore, CRISPR’s potential to modify vectors of diseases, such as mosquitoes, opens avenues for controlling vector-borne illnesses like malaria [46].

Conclusion

In conclusion, CRISPR technology has undeniably transformed the landscape of modern medicine, offering unprecedented precision and versatility for disease diagnosis and treatment. From revolutionizing diagnostics through tools such as SHERLOCK and DETECTR to enabling therapeutic breakthroughs for monogenic disorders, cancer, and infectious diseases, CRISPR's potential is vast. Emerging innovations, such as prime-editing and novel delivery systems, continue to refine its capabilities while expanding its applications. However, significant challenges, including off-target effects, immune responses, and accessibility, must be addressed to translate laboratory advancements into equitable, real-world solutions. As CRISPR technology advances, interdisciplinary collaboration among researchers, clinicians, ethicists, and policymakers. This collaborative effort will ensure that the profound benefits of CRISPR are realized responsibly and inclusively and will shape a future where precision medicine and global health advancements become a reality for all.