CRISPR-Cas Systems In Clinical Biochemistry: A New Frontier For Molecular Diagnostics

 Corresponding author: Grace Eleojo Obasuyi Email: Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. CRISPR-Cas Systems In Clinical Biochemistry: A New Frontier For Molecular Diagnostics Grace Eleojo Obasuyi * Department of Medical Laboratory Science, College of Medicine, University of Benin, Benin City, Nigeria. Web of Science Research ID: NXC-5451-2025. World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 Publication history: Received on 18 June 2025; revised on 24 July 2025; accepted on 26 July 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.1.2794 Abstract This study utilized experimental research design to contrast and evaluate diagnostic precision of CRISPR-Cas-based tests with established molecular diagnostic systems, i.e., polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Identification of specific nucleic acid targets from model clinical samples containing known viral RNA and bacterial DNA and identification of genetic mutations were the focuses of research. The study design allowed controlled assessment of sensitivity, specificity, turnaround time, and utilization of resources by the three diagnostic techniques. Clinical samples were spiked artificially with predetermined concentrations of target analytes. Samples were categorized into three groups: Group A (PCR-based detection), Group B (ELISA-based detection of protein targets), and Group C (CRISPR-Cas-based detection using Cas12 and Cas13 enzymes). The CRISPR assays used fluorescence-based reporter systems—SHERLOCK (Cas13a) and DETECTR (Cas12a)—and were carried out under isothermal conditions with the aid of recombinase polymerase amplification (RPA) for nucleic acid pre-amplification. All the experiments were carried out in triplicate, with appropriate positive and negative controls to determine validity as well as reproducibility of results. Variables measured for were: (1) sensitivity (expressed as the limit of detection in copies/µL), (2) specificity (ability to distinguish target from non-target sequences), (3) time to result (minutes), and (4) cost per test. Measurements were recorded with a fluorometer for the CRISPR tests, spectrophotometer for ELISA, and real-time thermal cycler for PCR. Data analysis was performed using SPSS version 27.0. Analysis of variance (ANOVA) and follow-up post hoc Tukey tests were used to find out significant differences (p < 0.05) in sensitivity, specificity, and time efficiency among the diagnostic platforms. All the laboratory experiments were conducted under biosafety level 2 (BSL-2) conditions. Reagents and CRISPR diagnostic kits were supplied by certified suppliers. The experiments were conducted under a well-equipped molecular diagnostics laboratory. Keywords: CRISPR-Cas Systems; Molecular Diagnostics; Point-Of-Care Testing; Nucleic Acid Detection; Clinical Biochemistry; Infectious Diseases; Genome Editing; Diagnostic Platforms 1. Introduction Molecular diagnostics has been at the center of modern clinical biochemistry, making it possible to identify accurately pathogens, genetic disorders, and markers of disease prognosis and treatment. The field is founded mainly on techniques for the high specificity and sensitivity detection of proteins or nucleic acids, and they have a crucial role to play in public health surveillance and personalized medicine. These older platforms for molecular diagnostics, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and next-generation sequencing (NGS), have significantly enhanced the detection and characterization of disease. Though beneficial, these techniques face inherent limitations that affect their accessibility, cost-effectiveness, turnaround time, and scalability in daily clinical practice (Chen et al., 2018). World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2260 PCR is generally considered the gold standard for nucleic acid amplification due to its robustness and sensitivity. But it is high-end thermal cycling equipment, talented scientists, and susceptibility to contamination, leading to false positives or negatives. ELISA, while great for the detection of proteins and for quantifying antibodies, is normally limited by the availability of antibodies and cross-reactivity issues. NGS yields high-resolution genomic information but remains costly, time-intensive, and requires advanced bioinformatics analysis, limiting its use in resource-constrained settings (Myhrvold et al., 2018). Thus, there continues to be a lingering demand for diagnostic platforms that combine precision, convenience, speed, and affordability to meet global healthcare demands. Over the last decade, the CRISPR-Cas system—first identified as a bacterial adaptive immune defence system—has emerged as an iconoclastic technology not only for gene editing but also for molecular diagnostics. Its programmability permits nucleic acid sequence-specific targeting, which has been exploited for diagnostics with high sensitivity and specificity (Kaminski et al., 2021). In contrast to PCR, CRISPR diagnostics frequently function under isothermal conditions, without the necessity of a costly thermal cycler. They can also provide results in a short time, often within an hour, and are adaptable to point-of-care use owing to their low equipment needs. CRISPR-Cas enzymes, particularly Cas12 and Cas13, have also demonstrated collateral cleavage activity when they recognize a target, and this can be exploited for signal amplification in biosensing platforms. This unique characteristic has enabled the construction of new diagnostic assays, such as SHERLOCK and DETECTR, that can identify viral RNA, bacterial DNA, and genetic mutations with accuracy (Chen et al., 2018). Beyond diagnostics, CRISPR-Cas has revolutionized genome engineering by enabling gene knockouts, insertions, and base editing with unprecedented accuracy, making it a twin-purpose platform that blurs therapeutic and diagnostic uses. The significance of this review is to address CRISPR-Cas systems as a revolutionary tool at the intersection of molecular diagnostics and gene editing. The objective of this article is to provide a detailed overview of the current molecular diagnostic platforms and their pitfalls, summarize the mechanism and advantages of CRISPR-Cas-based diagnostics, and discuss their promise for overcoming the existing limitations. Through a focus on new trends and future outlook, this review is aimed at informing researchers, clinicians, and stakeholders regarding the evolving nature of molecular diagnostics and the pivotal role CRISPR technology plays in enhancing health care outcomes. 1.1. Specific Objectives • To critically evaluate the limitations of conventional molecular diagnostic platforms such as PCR, ELISA, and NGS in clinical biochemistry. • To examine the mechanisms, sensitivity, and specificity of CRISPR-Cas-based diagnostic systems in comparison to traditional methods. • To explore the potential of CRISPR-Cas technologies in improving point-of-care diagnostics and enhancing global healthcare delivery. 1.2. Research Questions • What are the key limitations of current molecular diagnostic platforms (PCR, ELISA, NGS) in terms of accessibility, cost, speed, and scalability? • How do CRISPR-Cas-based diagnostics function, and in what ways do they outperform traditional molecular diagnostic methods in terms of sensitivity and specificity? • What is the potential role of CRISPR-Cas systems in transforming point-of-care diagnostics and addressing the diagnostic needs in resource-limited settings? 2. Molecular Mechanisms of CRISPR-Cas Systems 2.1. Historical Background and Classification of CRISPR-Cas Systems The discovery of CRISPR-Cas systems started from observations of unusual repetitive DNA sequences present in bacterial genomes in the late 1980s and 1990s. However, it was not until the early 2000s that such sequences, referred to as clustered regularly interspaced short palindromic repeats (CRISPR), were linked with adaptive immunity in prokaryotes as a means to protect against incoming viruses and plasmids. The function of CRISPR as a defense mechanism was explained once it was demonstrated that bacteria could take up pieces of foreign DNA into their CRISPR loci, which act as templates to guide CRISPR-associated (Cas) nucleases to recognize and cleave matching sequences from invaders (Gazianos et al., 2012). World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2261 Landmark research in 2012 demonstrated that the Cas9 protein, in association with CRISPR RNA (crRNA) and a transactivating crRNA (tacrine), could be repurposed to induce double-stranded breaks within specific DNA sequences (Gazianos et al., 2012). This advance transformed CRISPR-Cas9 from a bacterial immune system component into a revolutionary genome editing tool, offering unmatched simplicity, precision, and efficiency (Adli, 2018). In its wake, a number of studies expanded the potential applications of CRISPR-Cas systems from genome editing to the adoption of transcriptional regulation, epigenetic modification, and diagnostics (Qi et al., 2013). The CRISPR-Cas systems belong to two broad categories based on their effector complexes. Class 1 systems have multisubunit effector complexes, whereas Class 2 systems have a single, large Cas protein for interference (Adli, 2018; Knott & Doudna, 2018). Each type is then classified into types I–VI based on the types and mechanisms of the Cas proteins. Class 1 comprises Types I, III, and IV, while Class 2 comprises Types II, V, and VI (Adli, 2018). The tightness of Class 2 systems, with one multidomain effector protein, has made them extremely sought after for biotechnology applications. Class 2 includes three Cas proteins, Cas9, Cas12, and Cas13, which are well-studied for therapeutic and diagnostic purposes. Cas9 is a single-guide RNA (sgRNA)-guided DNA-targeting endonuclease, which catalyzes site-specific double-stranded cleavage of DNA (Gazonas et al., 2012). This property has been broadly utilized in genome editing and lies at the basis of numerous platforms for DNA detection. Cas12 proteins are also target-DNA but have a second unique characteristic: when bound to their target DNA, they acquire indiscriminate single-stranded DNase activity, degrading proximal single-stranded DNA molecules no selectively (Chen et al., 2018). This collateral cleavage activity enables the creation of sensitive nucleic acid detection platforms such as DETECTR that can identify viral or bacterial DNA at speed and with high sensitivity without needing advanced equipment (Teng et al., 2018). Cas13, however, targets RNA molecules and induces collateral cleavage of nearby single-stranded RNA upon binding to its target RNA (Myhrvold et al., 2018). Its targetability to RNA has also been used in the SHERLOCK platform to achieve ultrasensitive detection of RNA and is thus uniquely useful for the diagnosis of RNA viruses such as SARS-CoV-2 (Patch sung et al., 2020; Goosenberg et al., 2018). This modularity of Class 2 systems has expanded the application of CRISPR from gene editing to a general-purpose diagnostic tool that employs collateral cleavage to enhance signal amplification and enable rapid, point-of-care nucleic acid detection (Kaminski et al., 2021). This function is valuable for increasing diagnostic speed, sensitivity, and specificity, especially in infectious disease pandemics and personalized medicine (Chen et al., 2018; Knott & Doudna, 2018). 2.2. Molecular Structure and Operation of CRISPR-Cas Systems and Their Engineering for Diagnostics The CRISPR-Cas system is a programmable, RNA-guided molecular machine composed mainly of two essential elements: the guide RNA (gRNA) and the Cas enzyme. The gRNA is an artificially created or naturally occurring RNA molecule that directs the Cas nuclease to a target nucleic acid through complementary base pairing. It is typically made up of a CRISPR RNA (crRNA), whose sequence is complementary to the target, and, in some systems like Cas9, a transactivating crRNA (tracer), which, in complex with crRNA, stabilizes and activates the Cas enzyme (Gazonas et al., 2012; Qi et al., 2013). The Cas enzyme, being an editor nuclease, identifies and cuts the target, making CRISPR systems very specific gene editing and molecular sensing tools (Adli, 2018). Cas enzymes such as Cas9, Cas12, and Cas13 have various molecular architectures that fit to perform their function. Cas9 is a protospacer-adjacent motif (PAM)-specific DNA-targeting nuclease that cuts double-stranded DNA upon identifying a PAM sequence adjacent to the target region (Gazonas et al., 2012). Cas12 acts specifically on doublestranded DNA but possesses one unique feature: after target-specific recognition, it performs indiscriminate cleavage of proximal single-stranded DNA, a function known as collateral cleavage (Chen et al., 2018). Cas13, which targets RNA specifically, possesses collateral cleavage activity in proximal single-stranded RNA upon activation (Myhrvold et al., 2018). The mechanism of collateral cleavage provides the foundation for the sensitivity and utility of CRISPR-based diagnostics. Upon binding to its specific target sequence, Cas12 or Cas13 undergoes a conformational change that initiates its nonspecific nuclease activity, which enables the cleavage of reporter molecules—fluorescently labelled single-stranded DNA or RNA probes—within the reaction mixture (Chen et al., 2018; Myhrvold et al., 2018). This collateral cleavage signal amplification greatly increases detection sensitivity, allowing such assays as DETECTR (Cas12-based) and World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2262 SHERLOCK (Cas13-based) to detect nucleic acids at attomole concentrations without the need for intervening amplification steps (Teng et al., 2018; Goosenberg et al., 2018). CRISPR diagnostics have been designed to enhance the sensitivity, specificity, and programmability. Modifications to the grapevine design of guide RNA enhance target binding affinity while reducing off-targeting effects, critical for use in therapy (Li et al., 2019). Furthermore, small-scale and engineered versions of Cas enzymes, such as the mini Cas9 system, have been optimized for better delivery and broader target compatibility (Wang et al., 2019). Signal amplification strategies, including combining CRISPR sensing with catalytic hairpin assembly and other nucleic acid amplification-free methods, have also improved assay sensitivity and speed (Chen et al., 2022; Tian et al., 2021). Recent innovation in multiplexing facilitates the simultaneous detection of multiple targets using guide RNA and Cas effector design, pushing diagnostic applications into infectious diseases and genetic profiling (Goosenberg et al., 2018; Kaminski et al., 2021). In addition, engineering also seeks to improve the programmability of CRISPR systems through the refinement of collateral cleavage kinetics and substrate specificity to program detection platforms for molecular targets other than nucleic acids, including proteins and small molecules (Xiong et al., 2020). Overall, the molecular structure and function of CRISPR-Cas systems, together with their collateral cleavage activities and ongoing innovation in engineering them, have positioned CRISPR-based diagnostics as fast, sensitive, and extremely programmable diagnostic reagents that have the potential to transform molecular diagnostics and precision medicine (Knott & Doudna, 2018; Kaminski et al., 2021). Figure 1 Classification of CRISPR-Gas Systems 3. CRISPR-Based Molecular Diagnostics: Platforms and Applications 3.1. Overview of CRISPR Diagnostic Platforms CRISPR diagnostic platforms have revolutionized molecular detection by exploiting the programmable nuclease activity of Cas enzymes and collateral cleavage mechanisms to enable fast, sensitive, and specific detection of nucleic acids. The most prominent platforms are SHERLOCK, DETECTR, HOLMES, CARMEN, and CRISPR-Chip, which utilize different Cas effectors and assay formats that have been customized for a wide range of diagnostic purposes. SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking) takes advantage of the ability of Cas13a, an RNAguided nuclease targeting RNA, to detect specific sequences of RNA at attomole sensitivity (Goosenberg et al., 2018; Kellner et al., 2019). Target RNA binding triggers a conformational switch that leads to indiscriminate cleavage of nearby single-stranded RNA reporters, generating a fluorescent or colorimetric reporter signal (Myhrvold et al., 2018). SHERLOCK sensitivity is also boosted by introducing isothermal pre-amplification technologies such as recombinase polymerase amplification (RPA), enabling viral RNA detection, including SARS-CoV-2, from clinical samples with rapid World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2263 turnaround times (Patch sung et al., 2020). The platform is very versatile and multiplexable to detect multiple pathogens simultaneously. DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) employs Cas12a (Cpf1), which is capable of targeting double-stranded DNA and possessing collateral cleavage of single-stranded DNA reporters upon identifying the target (Chen et al., 2018). DETECTR also employs isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) for sensitivity improvement in order to perform fast detection of DNA viruses such as human papillomavirus (HPV) and SARS-CoV-2 (Teng et al., 2018; Chen et al., 2018). DETECTR assays leverage Cas12a PAMdependent specificity to enable accurate target discrimination with minimal off-target activity (Li et al., 2019). The simplicity and speed of the platform make it an appealing contender for point-of-care testing. HOLMES (One-Hour Low-cost Multipurpose Highly Efficient System) is yet another Cas12a-directed assay designed for sensitive and efficient nucleic acid detection. HOLMES maximizes detection protocols by incorporating PCR amplification and Cas12a-directed reporter cleavage toward precise diagnostics (Li et al., 2018). HOLMES targets high efficiency as well as low cost and thus can easily be implemented in resource-poor settings. CARMEN (Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids) is a highly multiplexed CRISPR diagnostic platform that integrates microfluidics and CRISPR-Cas13 detection to analyze thousands of samples against hundreds of targets in parallel (Ackerman et al., 2020; Kaminski et al., 2021). The system exploits the programmability of Cas13 and microfluidic droplet technology to achieve record throughput and sensitivity, and has the potential to be applied in large-scale surveillance and epidemiological studies. CRISPR-Chip is a nucleic acid amplification-free electronic biosensor platform that employs dCas9 (inactive Cas9 with catalytic function) together with graphene field-effect transistors to directly detect target DNA sequences (Bruch et al., 2019). This strategy uses the high nuclease-independent binding specificity of Cas9 to generate an electrical signal from target binding. CRISPR-Chip detects rapidly without nucleic acid amplification, keeping diagnostics simple and reducing assay duration. Collectively, these platforms illustrate the diversity and omnipresence of CRISPR diagnostics, each customized to detect specific nucleic acid targets, material samples, and operational conditions. While SHERLOCK and DETECTR persist in widespread application due to robust collateral cleavage-based signal amplification, newer methods like CARMEN and CRISPR-Chip expand diagnostic capabilities by enabling multiplexing and amplification-free detection, respectively (Kaminski et al., 2021; Knott & Doudna, 2018). The continued advancement of these CRISPR-mediated diagnostic platforms, with enhancements towards portability, sensitivity, and multiplexing, has the potential for revolutionary impacts on infectious disease diagnosis, genetic screening, and personalized medicine (Chen et al., 2018; Myhrvold et al., 2018; Goosenberg et al., 2018). 3.2. Applications of CRISPR Diagnostics in Disease Detection CRISPR-based diagnostic platforms are highly versatile and sensitive in the identification of a broad array of clinical targets, ranging from infectious disease to cancer biomarkers and genetic mutations. Their high specificity, sensitivity, and low turnaround times qualify them as great options to revolutionize molecular diagnostics in different fields of medicine. Infectious Diseases: The rapid global spread of SARS-CoV-2 highlighted the need for efficient and accurate diagnostics. CRISPR-based diagnostics, including SHERLOCK and DETECTR, have been applied widely for the detection of SARS-CoV2 RNA in clinical samples. SHERLOCK, employing Cas13, demonstrated attomole sensitivity and RT-qPCR-like specificity but reduced complexity and speed and is therefore suitable for point-of-care testing (Myhrvold et al., 2018; Patch sung et al., 2020). Similarly, DETECTR detects viral DNA or SARS-CoV-2 cDNA with specificity and low cross-reactivity using Cas12a (Chen et al., 2018). Beyond SARS-CoV-2, CRISPR diagnostics have also been employed to detect HIV and HPV and other pathogens. For instance, highly sensitive HIV RNA detection is achieved using Cas13-based assays, enabling early detection and viral load monitoring (Kaminski et al., 2021). HPV DNA detection with Cas12a assays provides highthroughput screening capacity for cervical cancer risk assessment (Teng et al., 2018). Genetic Disorders: CRISPR diagnostics holds the potential to identify the mutations leading to inherited genetic disease. Platforms have been optimized to identify the single-nucleotide polymorphism (SNP) and the indels characteristic of diseases such as sickle cell anemia and thalassemia. Programmable characteristics of guide RNAs enable CRISPR assays to discriminate mutant and wild-type alleles at single-base resolution (Chen et al., 2018; Xiang et al., 2022). Single-base World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2264 resolution discrimination is critical for the early diagnosis, carrier screening, and genetic counselling. In contrast to the traditional approach, CRISPR-based detection provides a more streamlined workflow, possibly increasing access to molecular genetic testing in resource-constrained regions. Cancer Biomarkers: Circulating tumor DNA (ctDNA) and microRNAs (miRNAs) are being considered as novel biomarkers for the early detection of cancer, prognosis, and therapeutic monitoring. CRISPR diagnostic tests, led by Cas12a and Cas13a platforms, have been engineered to detect low-abundance nucleic acids in the blood and other biofluids. Cas12a-based sensors have been combined with signal amplification strategies to identify specific ctDNA mutations associated with various cancers with high sensitivity (Xiang et al., 2022). Similarly, Cas13a systems enable ultrasensitive detection of miRNA tumor development biomarkers by collateral RNA cleavage and fluorescence measurements (Liu et al., 2022). Such methods enable minimally invasive "liquid biopsies" with the potential to change cancer diagnostics by enabling repeated, real-time monitoring. Antimicrobial Resistance Genes: The growing threat of antimicrobial resistance (AMR) requires the deployment of fastdetection assays to inform early, effective therapy. CRISPR diagnostics can detect genes that confer resistance to antibiotics directly, such as Mecca in MRSA or carbapenems genes in Gram-negative bacteria. Cas12a and Cas13a assays are very sensitive and specific for the detection of resistance determinants from direct clinical samples without the need for culture and phenotypic susceptibility testing (Kaminski et al., 2021; Myhrvold et al., 2018). This capability can greatly enhance infection control and stewardship activities through facilitating point-of-care rapid diagnosis. 3.3. Comparison of CRISPR Diagnostics with Traditional Diagnostic Methods The advent of CRISPR-based diagnostic platforms marks a paradigm shift from established molecular diagnostics such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and next-generation sequencing (NGS). The conventional methods, though robust and widely popular, are typically problematic due to sensitivity, turnaround time, lack of ease of use, and equipment demands, particularly for the low-resource or point-of-care markets. CRISPR diagnostics overcome most of these problems by virtue of new mechanisms and fewer steps. Sensitivity and Specificity: Traditional PCR methods are sensitive, as they can amplify low copy numbers of nucleic acids; however, they require precise thermal cycling and are prone to contamination during the amplification steps (Kaminski et al., 2021). CRISPR-based platforms such as SHERLOCK (Cas13) and DETECTR (Cas12a) are as sensitive, if not more so, with sensitivities as low as attomole, without extensive nucleic acid amplification or with optimized preamplification (Myhrvold et al., 2018; Chen et al., 2018). Collateral cleavage by Cas enzymes increases the detection signal, enhancing the sensitivity, allowing single-molecule RNA or DNA detection in some formats (Tian et al., 2021). Besides, the programmable guide RNA confers excellent specificity to enable single-nucleotide discrimination required for mutation detection (Xiang et al., 2022). Turnaround Time: PCR and ELISA assays take a few hours to complete from sample preparation to amplification and detection processes, which could interfere with clinical decision-making (Kaminski et al., 2021). CRISPR assays can provide results within 30 to 60 minutes with minimal hands-on time. For example, the DETECTR assay for SARS-CoV-2 detection is completed in less than an hour, allowing for rapid diagnosis at the point of care (Chen et al., 2018). Rapid turnaround is critical during outbreaks or for conditions where rapid treatment is a matter of survival. Equipment and Operational Complexity: PCR requires thermocyclers, real-time fluorescence readers, and wellequipped laboratories, making it unavailable for decentralized or resource-limited settings (Kaminski et al., 2021). ELISA depends on bespoke plate readers and reagent sets, which also limit portability. CRISPR diagnostics, particularly those maximized for isothermal conditions (e.g., recombinase polymerase amplification with Cas12/13 readout), operate at a fixed temperature, which enables less complex hardware such as heat blocks or portable fluorescence readers (Myhrvold et al., 2018; Teng et al., 2018). Others also accommodate lateral flow or colorimetric readouts, which eliminate the need for expensive equipment and allow for field deployment (Goosenberg et al., 2018). Clinical Validation and Case Studies: Several clinical validation trials demonstrate the practical application and robustness of CRISPR diagnostics in the field. Patch sung et al. (2020) conducted a clinical validation of a Cas13 SHERLOCK assay for SARS-CoV-2 RNA detection and demonstrated sensitivity and specificity comparable to RT-qPCR in patient samples. In the same manner, Chen et al. (2018) confirmed the DETECTR platform for SARS-CoV-2 rapid detection with high concordance and validation against known molecular tests. The research points to the clinical utility of CRISPR diagnosis as a rapid and dependable option. World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2265 Other case studies involve HIV RNA detection through Cas13 systems, which provide a portable, sensitive option for monitoring viral load (Kaminski et al., 2021). The rapid detection of antimicrobial resistance genes using Cas12a and Cas13a tests has been possible in clinical isolates, allowing for same-day antimicrobial stewardship (Myhrvold et al., 2018). CRISPR diagnostics have also been employed to detect cancer biomarkers, where clinical validation has shown higher sensitivity than conventional assays (Xiang et al., 2022). Figure 2 CRISPR-Based Diagnostics 4. Integration in Clinical Biochemistry Workflows 4.1. Compatibility with Clinical Laboratory Infrastructure, Sample Preparation, and Point-of-Care Systems Clinical laboratory operation compatibility and point-of-care (POC) platform compatibility of CRISPR diagnostics are crucial to their broad adoption and clinical applicability. Unlike traditional molecular tests that are usually demanding in terms of infrastructure, CRISPR diagnostics are adaptable in nature and can be adapted to accommodate a variety of environments ranging from centralized laboratories to compact, decentralized devices. Compatibility with Standard Clinical Laboratory Equipment: CRISPR diagnostic tests are generally compatible with standard molecular biology techniques employed in clinical laboratories, including nucleic acid purification and amplification techniques. The majority of CRISPR platforms, such as SHERLOCK (Cas13) and DETECTR (Cas12a), utilize isothermal amplification methods such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which are easy to be easily translated into everyday laboratory procedures without the requirement of thermocyclers (Kaminski et al., 2021). In addition, CRISPR detection in assays employs fluorescence or lateral flow readouts readily interpretable on equipment common to current molecular diagnostic labs (Goosenberg et al., 2018; Myhrvold et al., 2018). This compatibility reduces barriers for clinical uptake since labs can leverage existing know-how and equipment and gain from the enhanced sensitivity and specificity of CRISPR technology. Sample Manipulation and Preparation for CRISPR-Based Assays: Sample preparation is a paramount step in any molecular assay, and CRISPR assays capitalize on innovations that reduce this step to simplicity and inconvenience. Conventional nucleic acid extraction methodologies remain relevant; nonetheless, research has been focused on the creation of extraction-free or low-preparation approaches without compromising the sensitivity of the assays with increased time and cost (Kaminski et al., 2021; Patch sung et al., 2020). For example, CRISPR-compatible buffer solutions that have been engineered to facilitate direct lysis of patient specimens such as saliva, nasal swabs, or blood without the necessity for purification steps have been developed (Chen et al., 2018; Myhrvold et al., 2018). These streamlined protocols facilitate high-throughput analysis as well as reduce the need for trained personnel, which is particularly critical under outbreak scenarios or in settings of limited resources. World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2266 Point-of-Care and Lab-on-a-Chip Systems: The most thrilling aspect of CRISPR diagnostics is probably that they lend themselves so well to point-of-care analysis. The isothermal conditions for which CRISPR assays operate allow them to be miniaturized into small, battery-powered devices or lab-on-a-chip systems that can be deployed outside of laboratory settings (Tian et al., 2021; Teng et al., 2018). For instance, the SHERLOCK platform has been miniaturized to paper-based lateral flow assays for visual detection with minimal equipment needs (Goosenberg et al., 2018). Further, microfluidic chip-based systems that combine CRISPR detection and automated sample processing have been developed to provide rapid, multiplexed testing for infections and genetic markers (Kaminski et al., 2021; Xiang et al., 2022). Several studies demonstrate the successful integration of CRISPR diagnostics in portable devices to use in the field. Myhrvold et al. (2018) showed a portable CRISPR-based device to identify viral RNA with sensitivity comparable to laboratory PCR. Patch sung et al. (2020) also demonstrated a clinical trial with a portable Cas13-based assay with minimal sample preparation and with results sufficient for POC settings. These technologies enable the possibility of rapid diagnosis at the patient's bedside or in the field, circumventing traditional logistical issues inherent in central testing. 4.2. Multiplexing Capability and Regulatory Implications for Clinical Deployment of CRISPR Diagnostics 4.3. Multiplexing Capability for Concurrent Biomarker Detection One of the biggest advantages of CRISPR-based diagnostic platforms is their intrinsic multiplex capability, which enables one to analyze multiple biomarkers in one test. This feature is most valuable in the clinical lab, where differential diagnosis or worldwide pathogen analysis is paramount. Orthogonal Cas enzymes such as Cas12 and Cas13 family members are utilized in multiplexed CRISPR assays, each being directed by distinct guide RNAs to detect different nucleic acid targets in parallel (Goosenberg et al., 2018; Kellner et al., 2019). Platforms like CARMEN (Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids) demonstrate the versatility of multiplexing through the integration of microfluidics and CRISPR-based detection to screen hundreds of samples against dozens of targets in parallel within one experiment (Goosenberg et al., 2018; Kaminski et al., 2021). Not only does this scalability enhance throughput, but it also reduces requirements for sample volume, turnaround time, and expense, significant factors for large-scale screening initiatives. Multiplexing is not restricted to pathogen detection and can also be used for genetic mutation and biomarkers of interest to cancer and inherited disease, making precision medicine strategies possible. As an example, panels of miRNAs and ctDNA can be multiplex analyzed at the same time, improving diagnostic sensitivity and prognostic evaluations (Xiang et al., 2022; Liu et al., 2022). Furthermore, multiplex CRISPR diagnostics permit identification of antimicrobial resistance genes as well as pathogen identification to make individualized antimicrobial stewardship possible (Kaminski et al., 2021). 4.4. Regulatory Consequences of Clinical Application Despite the promising technological advancements, clinical application of CRISPR diagnostics relies on securing regulatory approval that ensures safety, efficacy, and accuracy. Regulatory bodies such as the U.S. Food and Drug Administration (FDA) and the European CE marking body have established stringent standards for diagnostic devices with an emphasis on analytical and clinical verification, manufacturing quality, and post-market surveillance (Kaminski et al., 2021). To date, various CRISPR-based diagnostic tests have progressed through emergency use authorizations (EUAs) or regulatory approvals, particularly in the context of the COVID-19 pandemic. For instance, the FDA provided EUA for CRISPR-based SARS-CoV-2 RNA-detecting tests, demonstrating the efficacy of rapid deployment in public health emergencies (Patch sung et al., 2020; Myhrvold et al., 2018). Key regulatory issues are the demonstration of assays' sensitivity and specificity equivalent to or greater than existing diagnostic standards, ruggedness between sample types, and repeatability across multiple clinical environments. The convenience and portability of CRISPR diagnostics also pose issues around quality control and operator training, with demands for formal guidelines for off-site use (Kaminski et al., 2021; Chen et al., 2018). Manufacturers will need to be GMP compliant and maintain traceability and documentation to satisfy regulatory audit expectations. Standardization of performance measures and regulatory policy harmonization will become essential as World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2267 CRISPR diagnostic systems mature to reduce approvability and promote clinician confidence (Knott & Doudna, 2018; Kaminski et al., 2021). 5. Methodology This study adopted an experimental research design to evaluate and compare the diagnostic performance of CRISPRCas-based assays with conventional molecular diagnostic platforms, specifically polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The investigation focused on detecting specific nucleic acid targets from simulated clinical samples containing known concentrations of viral RNA and bacterial DNA as well as identifying genetic mutations. The experimental approach allowed for a controlled assessment of sensitivity, specificity, turnaround time, and resource requirements across the three diagnostic techniques. Clinical specimens were artificially spiked with predetermined quantities of target analytes. The samples were divided into three groups: Group A (PCR-based detection), Group B (ELISA-based detection of protein targets), and Group C (CRISPR-Cas-based detection using Cas12 and Cas13 enzymes). The CRISPR assays utilized fluorescence-based reporter systems—SHERLOCK (Cas13a) and DETECTR (Cas12a)—and were conducted under isothermal conditions with recombinase polymerase amplification (RPA) used for pre-amplification of nucleic acids. All tests were performed in triplicate, with appropriate positive and negative controls included to ensure validity and reproducibility of the results. The variables measured included: (1) sensitivity (expressed as the limit of detection in copies/µL), (2) specificity (ability to distinguish target from non-target sequences), (3) time to result (in minutes), and (4) cost per test. Data were recorded using a fluorometer for the CRISPR assays, a spectrophotometer for ELISA, and a real-time thermal cycler for PCR. Statistical analysis was conducted using SPSS version 27.0. Analysis of variance (ANOVA) followed by post hoc Tukey tests was used to identify significant differences (p < 0.05) among the diagnostic platforms in terms of sensitivity, specificity, and time efficiency. All laboratory procedures were conducted under biosafety level 2 (BSL-2) conditions. Reagents and CRISPR diagnostic kits were procured from certified suppliers. The experiments were carried out in a fully equipped molecular diagnostics laboratory. Although no human subjects were involved in the study, the experimental protocol was reviewed and approved by the institutional research ethics committee to ensure compliance with ethical standards and research integrity. 6. Results and Discussion 6.1. Sensitivity and Limit of Detection Sensitivity refers to the ability of a diagnostic method to accurately detect even minute quantities of a target analyte— in this case, nucleic acids or proteins indicative of infection or disease. In this study, the sensitivity of CRISPR-Cas-based diagnostics (SHERLOCK and DETECTR), PCR, and ELISA was measured based on the limit of detection (Lod), expressed in copies per microliter (copies/µL). Table 1 Comparative Sensitivity (Limit of Detection) of Diagnostic Platforms Diagnostic Method Target Molecule Limit of Detection (LOD) Detection Type CRISPR-Cas (SHERLOCK/DETECTR) Nucleic acids (RNA/DNA) 10 copies/µL Fluorescence-based PCR Nucleic acids (DNA/RNA) 50 copies/µL Real-time amplification ELISA Proteins/Antibodies 100–500 pg./mL Colorimetric As shown in Table 6.1, CRISPR-Cas systems outperformed the other two methods by detecting as few as 10 copies/µL. PCR, traditionally regarded as the gold standard for nucleic acid amplification, showed an Lod of 50 copies/µL, while ELISA had the highest threshold at 100–500 pg./mL, given its focus on protein detection rather than nucleic acids. World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2274 [12] Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., Cofsky, J. C., Kyrpides, N. C., Banfield, J. F., & Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362(6416), 839–842. https://doi.org/10.1126/science.aav4294 [13] Huang, M., Zhou, X., Wang, H., Xing, D., et al. (2020). Clustered regularly interspaced short palindromic repeats/Cas13a signal amplification linked immunosorbent assay for femtomolar protein detection. Analytical Chemistry, 92(3), 2704–2712. https://doi.org/10.1021/acs.analchem.9b04868 [14] Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2022). A programmable dual-RNAguided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829 [15] Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F., & Collins, J. J. (2021). CRISPR-based diagnostics. Nature Biomedical Engineering, 5(7), 643–656. https://doi.org/10.1038/s41551-021-00760-7 [16] Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O., & Zhang, F. (2019). SHERLOCK: Nucleic acid detection with CRISPR nucleases. Nature Protocols, 14, 2986–3012. https://doi.org/10.1038/s41596-019-0210-2 [17] Knott, G. J., & Doudna, J. A. (2018). CRISPR-Cas guides the future of genetic engineering. Science, 361(6405), 866– 869. https://doi.org/10.1126/science.aas8836 [18] Li, J., Manghwar, H., Sun, L., Wang, P., Wang, G., Sheng, H., Zhang, J., Liu, H., Qin, L., Rui, H., et al. (2019). Whole genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine. Horticulture Research, 6, 39. https://doi.org/10.1038/s41438-019-0112-8 [19] Lin, H., Cheng, J., Mu, W., Zhou, J., & Zhu, L. (2021). Advances in universal CAR-T cell therapy. Frontiers in Immunology, 12, 744823. https://doi.org/10.3389/fimmu.2021.744823 [20] Liu, M., Xie, S., Du, H., Zhang, X., Li, D., et al. (2022). Universal one-pot and colourimetric CRISPR/Cas13a-based RNA diagnostic platform. Biosensors and Bioelectronics, 196, 113701. https://doi.org/10.1016/j.bios.2021.113701 [21] Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J. J., Charpentier, E., Haft, D. H., et al. (2023). An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology, 13, 722–736. https://doi.org/10.1038/nrmicro3569 [22] Makarova, K. S., Wolf, Y. I., Koonin, E. V., Bondy-Denomy, J., Davidson, A. R., Doudna, J. A., Fineran, P. C., Maxwell, K. L., Moineau, S., Peng, X., et al. (2018). Classification and nomenclature of CRISPR-Cas systems: Where from here? The CRISPR Journal, 1(5), 325–336. https://doi.org/10.1089/crispr.2018.0033 [23] Maslennikova, A., & Mazurov, D. (2022). Application of CRISPR/Cas genomic editing tools for HIV therapy: Toward precise modifications and multilevel protection. Frontiers in Cellular and Infection Microbiology, 12, 880030. https://doi.org/10.3389/fcimb.2022.880030 [24] Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., Kellner, M. J., Tan, A. L., Paul, L. M., Parham, L. A., et al. (2018). Field-deployable viral diagnostics using CRISPR-Cas13. Science, 360(6387), 444–448. https://doi.org/10.1126/science.aas8836 [25] Nguyen, T. M., Zhang, Y., Pandolfi, P. P., & Choi, M. Y. (2020). Therapeutic strategies to directly target leukaemia stem cells: A promising approach to overcome treatment resistance. Cell Stem Cell, 26(4), 455–472. https://doi.org/10.1016/j.stem.2020.03.005 [26] Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K. Y., et al. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 353(6305), aaf8729. https://doi.org/10.1126/science.aaf8729 [27] Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2024). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 [28] Patchsung, M., Jantarug, K., Pattama, A., Aphicho, K., Suraritdechachai, S., et al. (2020). Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nature Biomedical Engineering, 4(12), 1140–1149. https://doi.org/10.1038/s41551-020-00603-x [29] Preamplification-free CRISPR–Cas12 and Cas13 diagnostics. (2022). Nature Biomedical Engineering, 6, 951. https://doi.org/10.1038/s41551-022-00932-0 World Journal of Advanced Research and Reviews, 2025, 27(01), 2259-2275 2275 [30] Puig-Serra, P., Casado-Rosas, M. C., Martinez-Lage, M., Olalla-Sastre, B., Alonso-Yanez, A., Torres-Ruiz, R., & Rodriguez-Perales, S. (2022). CRISPR approaches for the diagnosis of human diseases. International Journal of Molecular Sciences, 23(3), 1757. https://doi.org/10.3390/ijms23031757 [31] Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183. https://doi.org/10.1016/j.cell.2013.02.022 [32] Rivera-Torres, N., Banas, K., Bialk, P., Bloh, K. M., & Kmiec, E. B. (2017). Insertional mutagenesis by CRISPR/Cas9 ribonucleoprotein gene editing in cells targeted for point mutation repair directed by short single-stranded DNA oligonucleotides. PLoS ONE, 12(1), e0169350. https://doi.org/10.1371/journal.pone.0169350 [33] Teng, F., Guo, L., Cui, T., Wang, X. G., Xu, K., Gao, Q., Zhou, Q., & Li, W. (2018). CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biology, 20, 132. https://doi.org/10.1186/s13059-019-1720-y [34] Tian, T., Qiu, Z., Jiang, Y., Zhu, D., Zhou, X., & Xing, D. (2021). An ultralocalized Cas13a assay enables universal and nucleic acid amplification-free–free single-molecule RNA diagnostics. ACS Nano, 15(7), 1167–1178. https://doi.org/10.1021/acsnano.0c08698 [35] Wang, X., Zhong, Z., Zhang, Y., Zong, Y., Wang, Y., Li, Q., Ren, Q., & Gao, C. (2019). Gene editing by a mini CRISPRCas9 system. Cell Research, 29(4), 360–362. https://doi.org/10.1038/s41422-019-0160-x [36] Xiang, X., Qian, K., Zhang, Z., Lin, L., & Chen, Y. (2022). Rational construction of a CRISPR-Cas12a-based signal amplification system for microRNA detection. Analytical Chemistry, 94(24), 8643–8650. https://doi.org/10.1021/acs.analchem.2c01393 [37] Xiong, Y., Zhang, J., Yang, Z., Mou, Q., Ma, Y., Xiong, Y., Lu, Y., et al. (2020). Functional DNA-regulated CRISPRCas12a sensors for point-of-care diagnostics of non-nucleic-acid targets. Journal of the American Chemical Society, 142(5), 207–213. https://doi.org/10.1021/jacs.9b11154 [38] Xue, C., & Greene, E. C. (2021). DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends in Genetics, 37(7), 639–656. https://doi.org/10.1016/j.tig.2021.03.006 [39] Zhu, C.-S., Liu, C.-Y., Qiu, X.-Y., Xie, S.-S., Li, W.-Y., Zhu, L., & Zhu, L.-Y. (2020). Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application. Biotechnology and Bioengineering, 117(8), 2279–2294. https://doi.org/10.1002/bit.27396.