Intracellular biosensors by functional nanomaterial-integrated CRISPR technologies for real-time molecular sensing

Abstract

CRISPR technology, originally developed as a genome-editing tool, has recently emerged as a powerful platform for intracellular biosensing. By harnessing the programmability and target specificity of CRISPR-associated proteins, such as Cas9, Cas12, and Cas13, researchers have engineered biosensors capable of detecting a wide array of intracellular signals, including nucleic acids, non-coding RNAs, and small-molecule metabolites. This review discusses the recent advancements in CRISPR-powered biosensors for real-time, dynamic monitoring of cellular processes and molecular events. Particular focus is given to the integration of nanotechnology, which plays a crucial role in enhancing the delivery efficiency, signal amplification, and sensor stability. Nanomaterials such as gold nanoparticles, quantum dots, DNA nanostructures, and upconversion nanoparticles have been strategically employed to improve the intracellular transport of CRISPR components, facilitate signal readouts, and enable multimodal sensing in complex cellular environments. Additionally, we delve into how CRISPR-nanotechnology hybrids can be adapted for multiplex analysis and single-cell resolution. This review also addresses the current challenges in intracellular biosensing, including precise delivery, biocompatibility, and long-term monitoring, and outlines future directions for the application of these systems in precision medicine, synthetic biology, and advanced therapeutic monitoring. Through the convergence of gene-editing systems and nanotechnology, CRISPR-based intracellular biosensors are anticipated to revolutionize next-generation diagnostic and therapeutic strategies.

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Min Yu Choi

Min Yu Choi is a graduate researcher in the School of Chemical Engineering at Jeonbuk National University. She received her bachelor's degree in chemical engineering in 2022 and is currently pursuing postgraduate research in nanobiosensors. Her work focuses on nanomaterial-based optical and electrochemical biosensors, including CRISPR-Cas12a-assisted SERS platforms, multiplex metal-enhanced fluorescence sensors, and electrochemical microneedle systems for real-time monitoring of disease-related biomarkers. She has co-authored several papers in international journals in the fields of biosensors and analytical chemistry.

Chenzhong Li

Prof. Chenzhong Li is the President's Distinguished Chair Professor of Biomedical Engineering at the School of Medicine, The Chinese University of Hong Kong (Shenzhen). He previously served as a research officer at the Biotechnology Research Institute of the National Research Council of Canada and held faculty appointments at Florida International University and Tulane University. Prof. Li is co-Editor-in-Chief of Biosensors and Bioelectronics and an associate editor of several leading journals; he is an elected fellow of the American Institute for Medical and Biological Engineering and the National Academy of Inventors. His research interests span biosensors, bioelectronics, lab-on-a-chip systems, point-of-care testing, and theranostic devices, and he has published more than 180 peer-reviewed papers and holds 18 international patents in biomedical sensing technologies.

Jin-Ha Choi

Prof. Jin-Ha Choi is an Associate Professor in the School of Chemical Engineering at Jeonbuk National University, where he leads the Nanomedical Engineering Laboratory. He obtained his PhD in Chemical and Biomolecular Engineering from Sogang University in 2014. His research focuses on nanotechnology-based biosensors and nanotherapeutics for early diagnosis and treatment of cancer and neurological diseases, with particular emphasis on CRISPR-based biosensing platforms, exosome-derived biomarkers, and 2D/3D biohybrid organoid- and organ-on-a-chip systems. He has authored numerous articles in international journals on biosensors, bioelectronic interfaces, and organoid-based disease models, and actively collaborates with domestic and overseas partners in the field of nanobioengineering.

Jeong-Woo Choi

Prof. Jeong-Woo Choi is a Loyola Distinguished Professor in the Department of Chemical & Biomolecular Engineering, Sogang University. He received a PhD at Rutgers University (USA), DEng at Tokyo Institute of Technology (Japan), and DBA at the University of Durham (UK). He was a visiting scientist in the ‘IBM Research-Almaden Lab’ (USA) and ‘Mitsubish Electronics Advanced Technology R&D Center’ (JP). He was a President of the ‘Korean Biochip Society’, a President of ‘Korean Society for Biotechnology and Bioengineering’, and an Editor-in-Chief of ‘Nano Convergence’ Journal. He is a fellow of ‘Korean Academy of Science and Technology’, and a fellow of ‘National Academy of Engineering of Korea’. He has published 499 articles in international journals including ‘Science’ and edited/authored 23 international books including ‘Handbook of Bioelectronics’. His research topics are mainly on nanobioelectronic device-based ‘Biosensor’, ‘Organoid-on-a-Chip’ and ‘Biohybrid Robot’.

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Introduction

Detection and imaging of biomolecules within living cells play a central role in elucidating the complex spatiotemporal distribution and dynamic changes in cellular components.¹ Such analyses extend beyond simple identification of molecular presence and contribute to the understanding of the functional mechanisms of intracellular molecular networks, including protein–nucleic acid interactions, signaling pathways, and metabolic fluxes. Direct molecular imaging of living cells minimizes artifacts that may arise from fixation or destructive treatments, thereby providing reliable data that more accurately reflect physiological conditions. Moreover, sensitive and precise monitoring of intracellular molecules enables real-time tracking of the expression patterns and dynamics of disease-associated biomarkers.^2,3 This capability can be applied to the early diagnosis and staging of diseases such as cancer, neurodegenerative disorders, and viral infections and provides critical evidence for evaluating drug responsiveness and optimizing therapeutic strategies.⁴ High-resolution and ultrasensitive biosensing technologies are gaining increasing importance as they are directly linked not only to clinical diagnostics but also to the advancement in personalized medicine.

Consequently, there has been a growing interest in systems capable of sensing and monitoring nucleic acids, proteins, and ions within the cellular environment, with particular attention given to clustered regularly interspaced short palindromic repeat (CRISPR)-based platforms that generate highly specific signals in response to even single-nucleotide variations. CRISPR-based genome-editing technologies have attracted significant attention for potential therapeutic applications, including the treatment of genetic disorders and cancer.⁵ Moreover, certain types of CRISPR exhibit collateral cleavage activity upon target sequence recognition, which has demonstrated significant potential in biosensing applications where the specific recognition of a target must generate a measurable signal.^6–8 In this context, CRISPR/Cas12a and Cas13a have been extensively used and have shown high detection accuracies for DNA and RNA targets, respectively.^9,10

Another notable advantage is their functionality at mild temperatures, suggesting the feasibility of their operation in living cells. These features highlight the potential of CRISPR technology as an alternative to PCR, which is regarded as the gold standard for nucleic acid detection. Although PCR offers high accuracy and is used in clinical diagnostics, it is limited by its complexity, lengthy processing time, and requirement for trained personnel. Moreover, PCR cannot capture dynamic changes in living systems and is prone to environmental interference during sample preparation.¹¹ Conversely, CRISPR systems can be flexibly integrated into intracellular biosensing for real-time and dynamic monitoring.^11,12

However, there are still limitations in delivery efficiency and stability in vivo, and it remains challenging to obtain sufficient signals in the complex biological environment.¹³ In addition, the intrinsic off-target effects of the CRISPR system, which refer to unintended recognition and cleavage of non-target sequences, restrict its performance in living cells.^14,15 Thus, various nanomaterial integration methods have been proposed to overcome these limitations. Various types of nanoparticles have been developed to enable the stable intracellular delivery of the CRISPR system, which is relatively large in size and susceptible to degradation.^16–18 Depending on the design, these nanoparticles may serve a single function or perform multiple roles, such as enhancing signal intensity through optical phenomena. Such improvements significantly increase the detection sensitivity of intracellular analytes.^19–22

This review first examines the mechanisms of CRISPR systems that have been actively investigated for biosensing, along with the unique characteristics associated with each CRISPR type. It then discusses the classes of biomolecules that are commonly targeted at the intracellular level and highlights the CRISPR-based applications developed for their detection. Particular attention is given to strategies addressing the delivery efficiency and stability of CRISPR systems, as well as the integration of nanomaterials designed to further enhance sensitivity. Finally, it outlines the current challenges and future perspectives of intracellular biosensing and monitoring using CRISPR technology.

CRISPR systems as intracellular biosensors

Mechanism and programmability of CRISPR-Cas systems

The CRISPR-Cas system originates from the bacterial immune response against invading nucleic acids and functions through a mechanism that recognizes and cleaves foreign sequences.^23,24 The activation and cleavage activity of CRISPR has been widely exploited as a powerful gene-editing technology in the life sciences.²⁵ CRISPR-based genome-editing technologies have been applied for transcriptional regulation, epigenome modification, genome-wide screening, and even chromosome imaging²⁶. Furthermore, certain CRISPR types possess trans-cleavage activity, in which nucleic acid recognition triggers the nonspecific cleavage of nearby single-stranded sequences. This property has enabled integration into various readout systems for biomarker sensing. Furthermore, CRISPR systems provide the programmability of the platform enables facile retargeting of different biomarkers simply by altering the crRNA sequence.³

CRISPR systems are highly diverse and are generally classified into two major classes: class 1 and class 2. Class 1 includes types I, III, and IV, whereas class 2 comprises types II, V, and VI.^27,28 Among these, Cas9, Cas12, and Cas13, which belong to class 2, have been extensively investigated as molecular diagnostic tools owing to their unique cleavage activities.^29–31

CRISPR-Cas9. CRISPR-Cas9 employs a chimeric single-guide RNA (sgRNA) formed by the fusion of crRNA and tracrRNA, which complexes with the Cas9 protein. Upon recognition of the protospacer-adjacent motif (PAM) sequence “NGG” within the target DNA, the HNH and RuvC nuclease domains of Cas9 are activated, resulting in cleavage of both the target and non-target strands of the bound double-stranded DNA.^32,33 CRISPR-Cas9 continues to serve as a mainstream genome-editing platform owing to its high versatility and broad applicability.³⁴ This reaction has been exploited in nucleic acid detection strategies that combine CRISPR/Cas9-mediated cleavage with nicking endonuclease-assisted amplification.²⁹ Furthermore, catalytically inactive Cas9 (dCas9), which lacks nuclease activity, has been engineered for use in target recognition or signal amplification through programmable binding to sgRNAs, protospacer sequences, or protein conjugates.

CRISPR-Cas13a. CRISPR-Cas13a is an RNA-guided endonuclease that specifically recognizes single-stranded RNA (ssRNA) targets and catalyzes the trans-cleavage of nearby ssRNA, usually without requiring a PAM sequence.²⁷ Upon complementary binding to the target RNA, the HEPN domain is activated and cleaves the RNA while simultaneously inducing collateral cleavage of the non-target RNA. This property enables signal amplification upon the recognition of a small number of targets, and, similar to other CRISPR systems, the crRNA sequence can be easily redesigned to detect different targets. When combined with isothermal amplification, Cas13-based platforms have demonstrated the ability to detect attomolar concentrations of DNA and RNA with single-molecule sensitivity and single-nucleotide resolution.³⁵ Moreover, distinct Cas13 family members exhibit different preferences for dinucleotide contexts in their trans-cleavage activity, allowing for simultaneous multiplexed detection.³⁰

However, RNA probes required for Cas13a-based sensing are more susceptible to degradation than DNA probes, which increases the risk of false-positive results.³⁶

CRISPR-Cas12a. CRISPR-Cas12a, an RNA-guided DNA endonuclease, is currently one of the most actively studied molecules. Cas12a binds to crRNA to recognize and cleave specific double-stranded DNA (dsDNA). The Cas12a enzyme recognizes the “TTTN” protospacer-adjacent motif (PAM) sequence, binds the activator site, and generates a PAM-distal dsDNA break with staggered 5′ and 3′ ends.^27,37 Following this cis-cleavage event, Cas12a exhibits collateral trans-cleavage activity in which adjacent single-stranded DNA (ssDNA) is randomly degraded. This target-dependent ssDNase activity has been harnessed in isothermal amplification-based assays to establish a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) platform, enabling rapid and highly specific DNA detection with attomolar sensitivity.³¹

Numerous studies have been conducted to expand the target range, enhance the cleavage efficiency, and reduce the off-target effects of Cas12a. First, it is well established that single-stranded DNA (ssDNA) can also activate Cas12a.³⁸ This property has allowed its application in the detection of various biomarkers without the need for a Protospacer Adjacent Motif (PAM) sequence.³⁹ Furthermore, the discovery that Cas12a can be activated by extending the 3′ terminus of short sequences with random sequences has increased the flexibility in designing activator strands. A method was also introduced to detect RNA targets directly without prior RNA amplification by utilizing the relatively stable Cas12a. This approach involves supplying short ssDNA or PAM-containing double-stranded DNA (dsDNA) to the seed region of the crRNA to enable RNA target detection at the crRNA terminus, although this still requires the addition of exogenous DNA.⁴⁰

Owing to their high target specificity and ability to generate measurable signals through collateral cleavage, CRISPR-Cas systems have been integrated with a broad range of reporter strategies and extensively studied in biosensing applications (Fig. 1).⁴¹

Fig. 1 Mechanisms of CRISPR-Cas systems. (a) Cas9 and sgRNA bind with target dsDNA, and the nuclease domains cleave the target strands. (b) Cas13a activated by target RNA and enables trans-cleavage to adjacent ssRNAs. (c) Cas12a and crRNA recognized target DNA and induced trans-cleavage to near ssDNAs. Reproduced from ref. 33 with permission from the Royal Society of Chemical, copyright 2023.

Types of intracellular targets

The intracellular environment contains a variety of biomolecules, some of which serve as biomarkers reflecting the state of the cell or disease. To discuss intracellular biosensing using CRISPR, it is important to understand which types of markers can be targeted in order to emphasize the necessity of intracellular monitoring.

Genomic DNAs. DNA is a central biomolecule that encodes genetic information and exerts a direct influence on cell fate and genomic stability through essential processes such as transcription, replication, damage, and repair. Structural alterations or sequence-level DNA abnormalities are closely associated with genomic instability, mutagenesis, chromosomal rearrangements, and structural variations. These events underlie diverse pathological conditions, including cancer, aging, and neurodegenerative disorders, underscoring the importance of directly observing DNA dynamics within living cells for both biological and medical research. Besides chromosomal DNA, atypical DNA elements such as extrachromosomal DNA (ecDNA) have also gained attention, particularly in tumor cells, where they contribute to dysregulated gene expression and drug resistance.⁴² Accordingly, technologies capable of high-resolution imaging and real-time monitoring of the spatiotemporal distribution and interactions of nuclear DNA can provide crucial insights into the mechanisms of disease development.⁴³ However, direct detection of specific DNA sequences in living cells, particularly in non-repetitive regions, remains a significant challenge. Traditional approaches are largely confined to fixed cells. Achieving selective and sequence-specific visualization of DNA under conditions that preserve cellular activity requires new principles and innovative imaging tools.⁴⁴

DNA methylation is an epigenetic modification involved in various cellular functions and is regarded as a potential biomarker for disease diagnosis and monitoring.⁴⁵ This modification primarily occurs at cytosine bases within CpG islands, where it prevents the binding of transcription factors and RNA polymerase to promoter regions, ultimately leading to transcriptional repression.⁴⁶ Aberrant DNA methylation and the resulting dysregulation of gene expression have been reported to be associated with various diseases, including cancer.⁴⁷ Monitoring DNA methylation at the single-cell level provides valuable insight into dynamic cellular states and enables the characterization of cell heterogeneity, which is often obscured in bulk analyses.⁴⁸

RNAs. RNA plays a pivotal role in regulating diverse biological processes, and the dysregulation of RNA expression and subcellular localization is recognized as a critical factor in the onset of various diseases. Consequently, the imaging of RNA within cells is essential for elucidating regulatory mechanisms and facilitating disease diagnosis, offering new insights into potential therapeutic strategies.⁴⁹

The localization and translation of mRNA impact cellular physiology at various levels. At the single-cell level, mRNA trafficking enables rapid and localized responses to both intracellular and extracellular signals, thereby affecting cell motility and differentiation. Therefore, it is crucial to analyze mRNA imaging from the intracellular to multicellular level, extending beyond cytoplasmic localization.⁵⁰

Among noncoding RNAs, microRNAs (miRNAs) are short RNAs that exert significant regulatory functions in numerous biological pathways. They are frequently upregulated in cancer and are implicated in tumorigenesis, rendering intracellular sensing indispensable.⁵¹ For instance, the aberrant expression of the well-known oncogenic biomarker miR-21 is commonly associated with tumor initiation and metastasis. Similarly, the overexpression of miRNA-196 and miRNA-106 has been reported as an indicator of cancer metastasis.¹⁹ However, miRNA biomarkers that are overexpressed in tumor cells often remain present at non-negligible levels in normal cells, rendering them susceptible to interference from extracellular signals. Thus, advanced imaging strategies with improved spatial resolution are required to enable the accurate detection of miRNAs, specifically within tumor cells.⁵² Circular RNAs (circRNAs), a class of noncoding RNAs, have been implicated in cancer initiation and progression through multiple regulatory mechanisms. Owing to their broad presence in human cells and their tissue specificity, circRNAs have emerged as valuable biomarkers for cancer diagnosis.⁵² Specifically, circRNAs are widely distributed in human cells and have been reported to participate in numerous malignancy-related processes, including cell cycle regulation, tumorigenesis, invasion, metastasis, apoptosis, and angiogenesis.⁵³ Long noncoding RNAs (lncRNAs), typically defined as transcripts longer than 200 nucleotides, regulate gene expression either through direct interactions with mRNAs in the cytoplasm or by modulating mRNA translation.⁵⁴ They have been shown to influence cell proliferation, invasion, and apoptosis, while imbalances in lncRNAs are associated with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and desiccation syndrome. Furthermore, lncRNAs are increasingly recognized as promising biomarkers for the early diagnosis of cancers.⁵⁵

Other molecules. In addition to nucleic acids, a wide range of biomolecules carry essential cellular information, and intracellular systems have been designed to target these diverse molecules. Among these, adenosine triphosphate (ATP) plays a fundamental role in cellular metabolism and is indispensable for physiological and pathological processes such as cell growth, inflammation,⁵¹ and transplant immunity.⁵⁶ This highlights the importance of monitoring the ATP levels in living systems. Interferon-γ (IFN-γ), primarily secreted by activated T cells and cytotoxic T lymphocytes (CTLs), is an effective immune checkpoint biomarker. Although liquid biopsy-based assays for circulating IFN-γ have been proposed, discrepancies often arise between systemic concentrations and those within the tumor microenvironment. This indicates that intracellular or localized measurement of IFN-γ is necessary to accurately reflect cytokine dynamics in tumor-associated immune responses.⁵⁷ Genomic instability caused by DNA damage is a key factor in many diseases. Cells use DNA repair enzymes such as flap endonuclease 1 (FEN1) and apurinic/apyrimidinic endonuclease 1 (APE1) to preserve their genomic integrity and stability.⁵⁸ Therefore, assessing the enzymatic activities of these DNA repair proteins can provide valuable insights into disease prevention, diagnostics, and prognosis.⁵⁹ Telomeres, repetitive DNA sequences at chromosome ends, play a critical role in maintaining chromosomal stability and regulating cellular senescence. This protective function is supported by telomerase, a reverse transcriptase that replenishes telomeric DNA and prevents its progressive shortening during replication. Although telomerase activity is typically absent in most normal somatic cells, it is reactivated in stem cells and highly expressed in cancer cells, making it an important biomarker for oncological studies.⁶⁰ Glutathione (GSH), a tripeptide and essential antioxidant, plays a central role in cellular defense by detoxifying reactive oxygen species (ROS), promoting antioxidant activity, and maintaining redox homeostasis. Accurate quantification of intracellular GSH levels is critical for assessing health status and enabling early detection of disease.⁶¹ Numerous small molecules serve as key targets for intracellular biosensing, enabling applications in disease diagnostics, cellular microenvironment monitoring, and immune response evaluation. The ability to detect diverse intracellular biomarkers while monitoring and modulating the intracellular environment underpins a broad spectrum of biological applications, including the regulation of cellular differentiation.⁶²

Nanotechnology-enabled CRISPR biosensing

The stable delivery of CRISPR into cells is a prerequisite for monitoring of various biomolecules in the intracellular environment. However, the delivery of CRISPR systems is generally hindered by the large size of Cas proteins and the inefficiency caused by endosomal and lysosomal degradation pathways.⁶³ Once delivered, CRISPR systems also face the risk of enzymatic or RNA degradation and interference owing to their highly complex intracellular environment.¹⁹ This limitation critically affects sensor performance, motivating ongoing research on nanomaterials that exhibit high biocompatibility, efficient loading capacity, stability, and specific catalytic activity.⁶⁴

Achieving stable delivery of the CRISPR system using nanoparticles also contributes to mitigating off-target effects. CRISPR inherently exhibits off-target activity, in which non-target sequences are mistakenly recognized, cleaved, and activated. To reduce such effects, extensive efforts have been made to engineer Cas proteins and guide RNAs (gRNAs).⁶⁵ Additionally, delivering CRISPR in the form of ribonucleoproteins (RNPs) or as a combination of mRNA and sgRNA can further improve specificity.⁶⁶ However, these approaches are often limited by challenges in delivery due to the large size and negative charge of the components. Therefore, technologies that protect CRISPR systems and enable their efficient delivery to the desired intracellular locations using nanoparticles are essential for successful in vivo biosensing applications.⁶⁷

Furthermore, when the CRISPR system recognizes targets and generates signals inside cells, the signal-to-noise ratio (SNR) is often compromised because of the complex intracellular environment. To address this issue, it is necessary to integrate various nanomaterials capable of enhancing optical readout signals. This chapter examines the commonly used nanocarriers for intracellular delivery and evaluates their potential for CRISPR/Cas system delivery. We also explored different types of nanomaterials designed to amplify the signal intensity within cells and reviewed how these materials have been integrated with CRISPR systems in ex vivo models.

Delivery enhancement of CRISPR components

For intracellular gene editing or target recognition, specific components should be delivered across the cell membrane and, in the case of gene editing, be taken up by the nucleus. The materials commonly employed for this purpose include metallic nanomaterials, DNA, and lipid-based biomolecules. These carriers should exhibit low cytotoxicity, allow the efficient loading of various probes for intracellular sensing, and maintain stability within the cellular environment. Optimizing nanocarriers for the delivery of the CRISPR system ensures its efficient activation, which has a significant impact on the specificity and sensitivity of biosensing.

Gold nanoparticles. Gold has been widely used for intracellular application owing to its biocompatibility, facile probe conjugation by thiol groups, structural versatility, and stability.⁶⁰ Choi et al. proposed a novel biomolecular electron controller (Biomoletron) based on AuNPs, which penetrated into cells to regulate the differentiation of SH-SY5Y cells into dopaminergic neurons. Traditional methods for controlling differentiation, such as micro-needle approaches, are generally disruptive and induce oxidative stress, which can inhibit cellular differentiation and lead to neuronal death due to membrane rupture. In contrast, Biomoletron utilizes biocompatible AuNP components to penetrate the cell membrane and deliver electrical stimuli, achieving controlled differentiation. Notably, even 48 hours post-internalization, cell viability remained above 98%.⁶⁸ Additionally, the delivery of nucleic acids into cells using AuNPs modified with polyethylene glycol (PEG), poly-L-lysine (PLL), or branched polyethyleneimine (bPEI) has been established for a considerable period.⁶⁹ Recently, AuNPs have been actively employed for simple nucleic acids and the delivery of complex gene-editing systems, and the intracellular delivery efficiency of AuNPs has been compared based on particle size, shape, and surface modifications. For example, the uptake efficiency of gold nanorods decreases with an increasing aspect ratio, whereas citrate-coated AuNPs of approximately 50 nm exhibit higher cellular uptake than smaller or larger particles.⁷⁰ Because AuNPs have long been considered carriers for intracellular delivery, various strategies have been proposed to enhance their performance, including surface modification, alterations in particle morphology, and conjugation with other nanomaterials, as well as to improve their stability for sustained intracellular monitoring. Li et al. demonstrated an approach for visualizing APE1 and FEN1 using gold nanoflares integrated with logic circuits, in which the nanoflares were composed of AuNPs densely coated with DNA strands to enhance cellular uptake and stability.⁵⁸ Song et al. developed nucleic acid-based gold nanorod (NAGNR) biosensors using gold nanorods as carriers and optimized the particle concentration to minimize cytotoxicity.⁷¹ In another study, concerns regarding the cytotoxicity of CTAB, which is used as a capping agent during gold nanorod synthesis, were addressed by ligand exchange witcitrate, enabling the real-time monitoring of intracellular miRNA dynamics during stem cell differentiation.⁷²

Thus, the potential of gold nanomaterials as delivery vectors for CRISPR systems has been demonstrated. Yuan et al. proposed a delivery strategy in which both the CRISPR system and the signal probe were immobilized onto AuNPs, thereby ensuring intracellular transport and effective activation of the sensing mechanism.⁷³ Mout et al. employed arginine-functionalized AuNPs to co-deliver CRISPR-Cas9 and sgRNA, thereby reducing the risk of immunogenic responses typically associated with plasmid-based delivery. This strategy enabled efficient gene editing within HeLa cells, in which the nanocomplexes persisted for up to 30 h without compromising cell viability (Fig. 2).⁷⁴

Fig. 2 Imaging of specific miRNA using CRISPR nanorobot. (a) Schematic of the detection system operated by CRISPR immobilized onto AuNPs. (b) Structure of a nanorobot for localization and enhancing detection efficiency. Reproduced from ref. 73 with permission from the American Chemical Society, copyright 2024.

DNA nanostructures. DNA nanostructures offer precise control over the spatial arrangement of signal-generating probes and structural features for material delivery, owing to their excellent self-assembly capabilities and high programmability.⁷⁵ They are also attractive nanocarriers owing to their high biocompatibility and minimal cytotoxicity.⁷⁶ Various structural designs have been tested to enhance cellular uptake and enable tumor-specific delivery,⁷⁶ and size optimization has been performed to avoid interactions with large intracellular molecules, such as nucleases and residual proteins.⁷⁷ Systems in which the proximity of two fluorescent probes is induced in the presence of target ions have also been reported, generating Förster resonance energy transfer (FRET) signals to monitor the targets.⁷⁸ This approach facilitates an optimal SNR during readout, ultimately improving the limit of detection.⁵² Furthermore, by incorporating specific sequences as single strands within the DNA nanostructures, they can act as catalysts to induce structural changes upon binding to target sequences, thereby enabling the detection of a wide range of intracellular species, including nucleic acids, proteins, and ions. Thus, DNA nanostructures are regarded as programmable platforms suitable for high-sensitivity imaging and molecular detection in living cells.⁷⁹

Accordingly, DNA nanostructures have also been explored for CRISPR system delivery. Tang et al. engineered a DNA origami template with PAM-rich regions to promote higher Cas9 loading and designed the origami structure with lockable features, enabling encapsulation of sgRNA/Cas9 complexes during intracellular delivery for more efficient transport.⁸⁰ Similarly, Xu et al. developed a DNA origami nanocage (DONC) capable of confining Cas9 RNPs within its internal space. This structure incorporates two chain locks that are selectively released by ATP and miR-21, allowing the complexes to be protected during delivery and subsequently released upon target recognition.⁸¹ Such DNA nanostructure-based delivery systems can be designed in diverse forms, and additional sequence designs can introduce multiple functionalities to ensure CRISPR activation at desired intracellular locations.

Nevertheless, there are limitations in achieving sufficient sensitivity for the detection of targets present at extremely low concentrations in living cells.⁵⁵

Lipid-based nanocarriers. Lipid nanoparticles (LNPs) typically consist of ionizable cationic lipids, polyethylene glycol (PEG) lipids, zwitterionic phospholipids, and cholesterol.⁸² Such structures not only facilitate efficient cellular uptake of LNPs but also improve their stability and enable the release of cargo into the cytoplasm.⁸³ LNP have been developed as efficient nanocarrier for various therapeutics and biomolecules in vivo. The numerous advantages of LNPs have made them highly attractive as next-generation delivery platforms.¹⁷ These advantages include precise tunability of delivery properties through lipid composition and structural modifications, receptor-mediated targeting specificity, scalability suitable for large-scale production, high gene modification efficiency, low immunogenicity, excellent biocompatibility, biodegradability, and sufficient payload capacity.⁸⁴ Additionally, the optimization of the LNP design and surface modification technologies enables tissue-specific targeting and prolongs circulation stability. Owing to these advantages, LNPs have emerged as promising candidates for the delivery of various nucleic acid-based therapeutics, including miRNAs, siRNAs, mRNA, CRISPR components, and small-molecule drugs.⁸⁵

However, when using the aforementioned ionizable cationic lipids, cargoes may not be effectively encapsulated at neutral pH due to the lack of charge. Wei et al. addressed this issue by incorporating additional cationic lipids that remain positively charged at neutral pH, preserving the tertiary structure and stability of Cas9/sgRNA RNPs during delivery. Using this LNP-based delivery system, they achieved selective gene editing in liver and lung tissues in mice.⁸⁶ Chen et al. employed a thermally stable, highly negatively charged iGeoCas9 mutant and optimized the LNP formulation to achieve tissue-specific therapeutic gene editing. These LNPs contain biodegradable and acid-degradable lipids, which help ensure the stable delivery of the CRISPR system in vivo.⁸⁷

Polymer-based nanoparticles. Polymeric nanoparticles represent a versatile category of polymer-derived nanomaterials encompassing structures, such as nanospheres and nanocapsules, generally ranging from 100 to 500 nm in diameter. Because of their straightforward preparation, adaptable architecture, tunable composition, and facile surface modification, they have garnered significant interest as carriers for the intracellular transport of diverse biomolecules and therapeutic agents⁸⁸. In general, such polymers include poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), chitosan, gelatin, poly(dimethylaminoethyl methacrylate) (PDMAEMA), and poly(trimethylaminoethyl methacrylate) (PTMAEMA), and hydrophilic polymers such as nucleic acids or polyethylene glycol (PEG) are also employed on the surface. PEI is one of the most widely used polycations because it induces endosomal rupture and facilitates the release of delivered components into the cytoplasm, thereby enhancing transfection efficiency. However, because of the cytotoxicity of high-molecular-weight PEI, ketalized PEI has been proposed as a strategy to reduce its toxicity. Deng et al. encapsulated the photosensitizer Ce6 and the CRISPR/Cas9 system within polymeric micelle nanoparticles (NTA-NPs) while incorporating cationic polymers to neutralize the negative charge. Inside cells, Ce6 generates reactive oxygen species (ROS) upon near-infrared irradiation (NIR), leading to lysosomal membrane disruption and subsequent release of the delivered components. CRISPR is released in response to glutathione (GSH), enabling the detection of Nrf2, an antioxidant regulator.⁸⁹

2D nanosheets. Two-dimensional (2D) nanomaterials are attractive materials for biosensors because of their unique physicochemical properties⁹⁰, and their high surface-to-volume ratios further highlight their potential as efficient drug delivery platforms.⁹¹ It also exhibits strong binding affinity toward ssDNA probes while showing negligible interaction with dsDNA, thereby providing a convenient platform for the intracellular delivery of target-recognition probes.⁹² Graphene oxide (GO) is widely used owing to its excellent aqueous dispersibility, biocompatibility, tunable surface chemistry, and low toxicity.⁹³ However, its structural instability may cause cellular damage during the delivery process; thus, it is often integrated with polymeric matrices to enhance its biocompatibility.

Signal amplification and readout platforms

Optical readout methods convert biological interactions into optical signals⁹⁴ and are widely employed for rapid and straightforward sensing and monitoring owing to non-ionizing radiation and high resolution.^95,96 Various nanomaterials with unique optical surface properties can further enhance signals and reduce background noise, thereby enabling sensitive detection. Representative optical detection methods used in intracellular sensing include fluorescence and Raman spectroscopy.

Fluorescence readout and materials. Fluorescent materials are excited at specific wavelengths and emit photons that exhibit distinct spectral peaks depending on the material. Their high absorption cross-section, quantum yield, specificity, and biocompatibility make them promising candidates for biosensing.⁹⁷ The development of various fluorescent probes, such as quantum dots (QDs) and upconversion nanoparticles (UCNPs), allows the selection of appropriate particles to maximize sensing performance.

QDs are nanocrystalline semiconductors that exhibit quantum confinement effects and provide unique optoelectronic properties.⁹⁸ They typically exhibit high quantum efficiency, strong photostability, broad excitation ranges, and narrow emission peaks.^99,100 A key advantage of QDs is that their emission wavelengths can be finely tuned by controlling the particle size and composition, covering the visible to NIR spectrum.¹⁰¹ This NIR window reduces the light scattering in biological tissues, thereby improving the resolution.⁹⁶ Another NIR-compatible fluorescent material, upconversion nanoparticles, sequentially absorb NIR photons and emit a single photon of higher energy, typically in the visible region.¹⁰² UCNPs offer adjustable size, low fluorescence background, deep tissue penetration, low cytotoxicity, and strong photochemical stability.^103,104 By adjusting the type and concentration of dopants, photophysical properties such as the emission wavelength, intensity, and lifetime can be tailored, enabling the design of detection systems for specific biological targets.^105–108 UCNPs absorbing 980 nm NIR energy can be further modified with Nd³⁺-doped shells to respond to a more tissue-compatible 808 nm laser. Gong et al. employed UCNPs as carriers and activators of DNA to analyze miR-21 in living cells and designed peanut-shaped UCNPs for efficient tumor cell delivery. In this study, the fabricated PUCNPs-NH₂/PEG-ZL-DNA successfully detected targets in cells under 808 nm light.¹¹

Surface-enhanced resonance scattering. Raman spectroscopy analyzes the intrinsic molecular signals based on the scattering of incident light.¹⁰⁹ Despite their high selectivity and potential for biosensing, their intrinsic Raman signals are weak. Surface-enhanced Raman scattering (SERS) overcomes this limitation by significantly amplifying Raman signals through electromagnetic (EM) mechanisms induced by the localized surface plasmon resonance of nanoparticles and chemical mechanisms (CM) related to charge transfer.¹¹⁰ Because EM is generally considered the primary amplification mechanism, nanostructures with gaps or junctions are often designed to create hotspots.¹¹¹ Uniform distribution of hotspots is crucial for SERS-based sensing and has prompted the development of various SERS nanotags. Li et al. fabricated core–shell Au@Ag–Au SERS nanotags to generate hotspots using single particles.¹¹² Raman reporters were positioned inside the hollow gaps and on the outer surface of the nanotags to form monolayers. This design leveraged the high stability of gold against oxidation and the strong molar absorption of silver to achieve uniform SERS enhancement. Choi et al. developed an SERS-active graphene oxide/periodic triangular Au nanoflower (TANF) array functionalized with Raman probe-conjugated AuNPs (RAuNPs) to sensitively detect multiple viral DNAs.¹¹³ In this study, the Au nanoflower substrate was triangular and surface-roughened to maximize the EM amplification. Additionally, DNA-conjugated RAuNPs on the substrate produced greater SERS enhancement than the Raman probe alone (Fig. 3).

Fig. 3 Schematic of RAuNPs immobilized GO/TANF array. Reproduced from ref. 113 with permission of the American Chemistry Society, copyright 2021.

Nanomaterials for enhancing signal intensity. Both readout methods generate target-dependent signals within cells and have been employed to detect intracellular drugs, biomarkers, and metabolites. However, achieving sufficient sensitivity requires additional elements to enhance the signal intensity. Plasmonic nanomaterials have been widely used for this purpose. Noble metals such as gold and silver are the most commonly used plasmonic materials because of their chemical stability, biocompatibility, and excellent electrical properties.^114–117 Gold nanostructures, including nanorods, nanostars, and nanoclusters, exhibit tunable localized surface plasmon resonance (LSPR) features.^118–120 Due to these unique surface properties, gold can induce the phenomenon of metal-enhanced fluorescence (MEF), which markedly amplifies fluorescence signals located at an optimal distance of approximately 7 nm from its surface¹²¹. Although the effective distance and degree of enhancement vary depending on the system, gold nanostructures can be fabricated in diverse forms and are easily combined with other components, allowing MEF to be applied in various configurations. For example, a sensing system utilizing hybrid nanorods composed of gold and magnetic nickel has been reported, enabling exosome separation, enrichment, and sensitive miRNA detection via MEF¹²².

Notably, several studies have demonstrated the use of MEF for intracellular biomarker detection. Choi et al. sensitively detected caspase-3, a biomarker for various diseases, inside cells by dual-conjugating AuNPs with fluorophore via a peptide and single-stranded DNA (ssDNA). Verification of this system in MCF-7 cells showed that it did not affect cell viability and produced strong fluorescence emission even at low intracellular target concentrations.¹²³ Gold nanorods conjugated with fluorescently labeled nucleic acid probes can be delivered into cells in a quenched state; upon target binding, probe unfolding induces fluorescence amplification through MEF, enabling intracellular target detection.⁷¹ Similarly, molecular beacons conjugated to gold nanorods have been delivered into stem cells to monitor miR-124 in real time, allowing for the observation of neuronal differentiation in human iPSC-derived neural stem cells.⁷² Silver nanomaterials provide higher plasmonic frequencies and stronger MEF effects than gold nanomaterials, making them suitable for ultrasensitive single-molecule detection. Their performance can be further enhanced through the structural engineering of nanocubes, nanowires, or nanoplatelets, although surface stabilization often requires protective coatings.^124–126

GO, a carbon-based nanomaterial, possesses intrinsic properties such as flexibility and biocompatibility, which have made it widely utilized in the development of biosensors.^127–129 The oxygen-containing functional groups of GO, including epoxides, hydroxyls, and carboxyls, confer hydrophilicity, negative surface charge, and chemical versatility, enabling interactions with biomolecules via electrostatic forces, hydrogen bonding, and π–π stacking.^130–132 Moreover, the amphiphilic, nanoscopic, and high surface area properties of GO promote cell adhesion, enhancing cellular attachment in substrate-based differentiation assays and contributing to improved sensor stability.¹³³ These properties also allow efficient immobilization of multiple biorecognition elements on a single sheet, facilitating multiplexed detection and preconcentration of low-abundance targets from complex samples.^134,135 Surface passivation strategies further reduce nonspecific adsorption, enhancing selectivity. These properties make GO an effective platform for simultaneous detection of multiple biomarkers, which is particularly valuable in analyzing complex disease states.^135,136 Moreover, GO can quench fluorescence through FRET, thereby enabling highly sensitive detection in fluorescence-based sensing applications.

These materials can be applied in combinations of two or more components to enhance sensitivity while maintaining stability or to generate hotspots that maximize the signal output. Mahani et al. developed a quenching-based fluorescent probe with high selectivity, sensitivity, and low toxicity by combining AuNPs with sulfhydryl-functionalized Cu-doped carbon quantum dots (S-Cu@CQD) and successfully imaged GSH in breast cancer cells.⁶¹ Plasmonic nanoassemblies utilizing hotspots induced by the simultaneous use of AuNPs and AuNRs enabled the sensitive detection of telomerase (TE) and miR-21 within cells, facilitating early cancer detection. In addition to combining two distinct materials, hotspot generation has also been achieved through core–shell particle designs. Xu et al. employed Au@4MBN@AuNPs, in which the Raman reporter molecule 4MBN was inserted between the metal core and the shell while using ROX particles for signal generation, significantly enhancing the ROX Raman signals and improving the accuracy and reliability of quantification (Fig. 4).⁴

Fig. 4 Optimization of the distance between AuNPs and fluorescence. (a) Schematic of the MEF phenomenon depending on the distance from AuNP surface. (b) Fluorescence intensity data for MEF verification based on the length of sequences. Reproduced from ref. 121 with permission of the American Chemical Society, copyright 2021.

Applications in live-cell and single-cell biosensing

Multiplexed analysis of intracellular targets

Assessing multiple variables simultaneously within a single cell is considered the key to understanding complex cellular functions.¹³⁷ Wang et al. developed a CRISPR/Cas9 system-based fluorescent probe for chromatin imaging of living cells. By labeling sgRNA with a fluorophore, they demonstrated the ability to image multiple targets simultaneously using different colors.¹³⁸ Extensive efforts have been devoted to imaging RNA within living cells because this approach enables a deeper understanding of cellular functions and provides new insights into disease therapy. Conventional RNA imaging strategies often require multiple fluorescent tags that may perturb normal cellular physiology. Jia et al. developed an RNA-imaging platform based on the dCas12a orthogonal system by combining a crRNA-based conformational switch and a controllable CRISPR activation (CRISPRa) system. This method enables simultaneous detection of cancer-related HER2 and survivin mRNAs in living cells.⁴⁹ Ke et al. developed a hybrid-engineered nuclease, deactivated AsCas12a (dAsCas12a), and hairpin-structured crRNA (h-CAP) to enhance the specificity and efficiency of Cas12a, enabling multiplex gene expression regulation in human cells.¹³⁹ Shen et al. established a CRISPR-Cas13a platform combined with T7 transcriptional amplification for simultaneous detection of FEN1 and APE1 using an AND logic gate. FEN1 is the central enzyme in the base excision repair (BER) pathway, whereas APE1 initiates downstream repair processes. Concurrent detection of both enzymes reduces false-positive errors in tumor cell identification.⁵⁹

Disease biomarker sensing in living cells

Recently, CRISPR has been frequently applied for the sensitive detection and imaging of cancer biomarkers within the intracellular environment, often for visualizing the tumor microenvironment (TME). Intracellular RNA targeting may cause non-specific cleavage events that can induce cytotoxicity. Chen et al. addressed this issue by engineering a modified CRISPR-Cas12a system and introducing artificial bubble structures into the seed region to eliminate the PAM sequence dependency on target recognition and cis-cleavage activity. This optimized system was validated for telomerase, ATP, and miR-21 detection. Sufficient trans-cleavage activity in living cells was confirmed using confocal laser scanning microscopy combined with a fluorescence-quencher reporter. Delivery was achieved using a commercial transfection reagent without any assistance.⁵¹ Considering the complexity of immune responses and the heterogeneity of the tumor microenvironment, Liu et al. designed a CRISPR/Cas12a-based system for sensitive detection of IFN-γ. Conventional antibody-based probes are often limited by background interference and poor tumor penetration. To overcome these limitations, the system incorporates an NIR photoactivatable aptamer, a CRISPR module, and UCNPs functionalized with cationic polymers for electrostatic assembly and intracellular delivery. The CRISPR system followed an AND logic design, being activated only in the presence of both light and IFN-γ, which enabled quantitative in vivo imaging of exogenous IFN-γ in a PBMC-engrafted mouse model.⁵⁷ Zhang et al. reported a fluorescent CRISPR (fCRISPR) system that enables multicolor genome imaging. This system employs fluorogenic proteins intrinsically unstable owing to a peptide-derived degron domain (tDeg), leading to rapid degradation. Binding to a specific RNA hairpin stabilizes proteins, allowing fluorescence emission and dynamic RNA visualization.⁴⁴

Moreover, for the efficient operation of CRISPR systems within living cells, sufficient metal ions, such as Mg²⁺, Mn²⁺, and Co²⁺, are essential to support trans-cleavage activity. Two-dimensional nanosheet-based nanomaterials have also been used for CRISPR-Cas12a delivery. Liu et al. used cobalt oxyhydroxide (CoOOH) nanosheets, which are positively-charged layered materials, to detect intracellular miR-21. In the absence of ascorbic acid (AA), steric hindrance prevents Cas12a activation; however, intracellular AA reduces CoOOH to Co²⁺, relieving the steric blockade. Co²⁺ ions further act as cofactors to enhance Cas12a catalytic activity, thereby improving the detection efficiency.¹⁹ Similarly, Wang et al. synthesized ultrathin MnO₂ nanosheets capable of binding to CRISPR-Cas12a components through π–π interactions. With their large surface area, excellent biocompatibility, and low cytotoxicity, MnO₂ nanosheets are suitable for biosensing applications. Inside cells, the nanosheets are reductively degraded by endogenous biomolecules, releasing the CRISPR system for target detection.¹⁴⁰ MnO₂ nanosheets are reduced by GSH to produce Mn²⁺, which in turn enhances the trans-cleavage activity of CRISPR/Cas12a, thereby improving sensitivity (Fig. 5).¹⁴¹

Fig. 5 Schematic of live-cell biosensing analysis of mRNA using MnO₂ as the carrier and accelerator of CRISPR/Cas12a. This image reproduced from Wang et al.¹⁴⁰ and licensed under a Creative Commons Attribution 3.0 Unported Licence. Copyright 2022, Royal Society of Chemistry.

Single-cell CRISPR biosensing

Real-time high-resolution imaging is essential for understanding the diverse dynamic behaviors of individual RNA molecules in a single cell. However, single-molecule live-cell imaging technologies capable of tracking RNA and DNA at a single-molecule resolution enable the investigation of biological phenomena that are difficult to capture through bulk analysis. To address this, research has been conducted using single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), which combines the CRISPR-Csm complex with multiplexed gRNAs. When applied to RNA labeling in cells, this approach provides sufficient signal intensity while reducing the high background noise that commonly occurs in conventional Cas system-based methods. This system successfully tracks individual native NOTCH2 and MAP1B transcripts in living cells.¹⁴²

Another study developed a CRISPR-mediated fluorescence in situ hybridization amplification (CRISPR FISHer) system to image specific DNA sequences in live cells. This method was used to record the movements of extrachromosomal circular DNA (eccDNA) and invading DNA, enabling the analysis of dynamic behavioral differences between the chromosomal and extrachromosomal loci.⁴³ This study enabled real-time imaging based on Cas9, allowing a deeper understanding of biological events within living cells. Furthermore, by facilitating the investigation of invading DNA replication and innate immune responses at the single-cell level, it holds potential to aid biomedical diagnostics and clinical therapeutics.

scCLEAN was developed to enhance the biological resolution of low-abundance transcripts. This system increases the complexity of lowly expressed molecules, thereby improving the sensitivity. Using this approach, scCLEAN enables the discrimination of subtle transitional states within a homogeneous cell population, highlighting its potential to advance single-cell methodologies.¹⁴³ This study achieved enhanced resolution of biological signals and provides a promising approach for advancing single-cell methodologies.

Perturb-FISH combines CRISPR perturbation with in situ gRNA detection, providing simultaneous spatial transcriptomic information at the single-cell level and revealing the molecular changes associated with specific genetic perturbations.¹⁴⁴

CRISPR-based single-cell detection and imaging approaches have made it possible to monitor the dynamic behavior of RNAs, DNAs, and other biomolecules sensitively and precisely, thus serving as powerful tools for advancing our understanding of complex biological processes within individual cells.

Challenges and future perspectives

The integration of CRISPR/Cas systems with nanomaterial-based biosensors has ushered in a new era of intracellular diagnostics and therapeutic monitoring. However, several challenges remain to be addressed to fully realize their potential.

Achieving precise delivery of CRISPR components to target cells remains a critical hurdle. Recent advancements in nanomaterial engineering have shown promise in enhancing delivery efficiency and specificity. These innovations aim to minimize off-target effects and improve therapeutic outcomes.

Biocompatibility of nanomaterials is important for their clinical applications. Ongoing research focuses on optimizing surface modifications to reduce immunogenic responses and enhance biocompatibility. The dynamic nature of the cellular environment poses challenges for the stability of biosensors. Strategies to improve sensor longevity include the development of robust nanomaterial coatings and the incorporation of protective biomolecular layers to shield the sensors from enzymatic degradation and environmental fluctuations. Enhancing the SNR is crucial to biosensor sensitivity. The path to clinical applications involves navigating complex regulatory landscapes. Standardization of fabrication processes, rigorous safety evaluations, and alignment with regulatory frameworks are essential steps. Additionally, the scalability of production methods is necessary to meet clinical demands.

Strategies to address these limitations are currently being actively explored. For instance, recent studies have employed nucleus-specific photoactivatable CRISPR system to monitor metal ions in vivo, and further incorporated an AND logic gate strategy to achieve more specific imaging.¹⁴⁵ Enabling such precise spatiotemporal control contributes to accurate monitoring of the target. Moreover, the integration of CRISPR/Cas-based biosensors into wearable and implantable devices is a promising strategy for continuous health monitoring. Recent developments in microfluidic systems and flexible electronics have facilitated the creation of noninvasive real-time biosensing platforms for personalized healthcare. CRISPR/Cas-based biosensors have the potential to revolutionize personalized medicine by enabling the real-time monitoring of therapeutic targets and disease biomarkers. This capability allows for tailored treatment strategies and timely adjustments to therapeutic regimens. The application of artificial intelligence and machine learning algorithms to biosensor data can enhance the data interpretation and decision-making processes. These technologies enable the analysis of complex datasets, leading to improved diagnostic accuracy and predictive modeling in clinical settings.

Conclusions

Recent developments in CRISPR-based intracellular biosensing, combined with advanced nanomaterials, have significantly improved the sensitivity, specificity, and versatility of molecular detection. Gold and lipid nanoparticles, DNA nanostructures, and smart plasmonic assemblies have enabled the efficient delivery of CRISPR components into live cells, while minimizing cytotoxicity and off-target effects. Concurrently, the integration of optical readouts such as fluorescence and Raman spectroscopy with plasmonic enhancement strategies has enhanced SNR, allowing real-time monitoring of diverse cellular analytes. These advancements collectively establish a robust foundation for next-generation diagnostic and therapeutic applications, enabling personalized and responsive healthcare solutions that leverage CRISPR-nanotechnology biosensing.

Author contributions

Conceptualization, M. C., J-H. C., and J-W. C.; investigation, M. C. and C. L.; writing – original draft, M. C. and C. L.; writing – review and editing, M. C. and J-H. C.; supervision, J-H. C. and J-W. C.; project administration, J-H. C. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(RS-2024-00344633), and by GRDC Cooperative Hub through the National Research Foundation of Korea funded by the Ministry of Science and ICT (Grant number RS-2023-00259341), and by National R&D Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (NRF-2022M3H4A1A01005271).

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Footnote

† The authors equally contributed to this work.

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