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Advances in CRISPR Based Biosensing Strategies for Cancer Diagnosis

  • Qi-Ling Yang,
  • Xin-Yi Zhong,
  • Lin-Min Zhong,
  • Hui Zhang and
  • Shan-Wen Hu* 
Cancer Screening and Prevention   2024;3(1):61-71

doi: 10.14218/CSP.2023.00026

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Revised:

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Citation: Yang QL, Zhong XY, Zhong LM, Zhang H, Hu SW. Advances in CRISPR Based Biosensing Strategies for Cancer Diagnosis. Cancer Screen Prev. 2024;3(1):61-71. doi: 10.14218/CSP.2023.00026.

Abstract

With the development of CRISPR/Cas technology, its application in tumor biosensing and diagnosis is becoming increasingly widespread. CRISPR/Cas-based platforms can enable sensitive detection of tumor-related targets through programmable target recognition, cleavage, and signal amplification. However, challenges remain in translating these platforms into routine clinical diagnosis. This review focuses on CRISPR/Cas-based biosensing strategies for cancer diagnosis, including the detection of tumor-derived exosomes, circulating tumor DNA, circulating tumor cells, and tumor biomarkers. These approaches combine CRISPR/Cas systems with aptamers, nucleic acid amplification, electrochemical, fluorescent, and other sensing strategies to facilitate the detection of low-abundance tumor-associated targets. Related CRISPR applications for investigating the tumor microenvironment are also briefly discussed. Current studies demonstrate the versatility and analytical sensitivity of CRISPR/Cas-based biosensing; however, further improvement in assay performance and clinical validation is needed before these strategies can be routinely applied to cancer screening or diagnosis.

Keywords

CRISPR/Cas biosensing, CRISPR/Cas, Exosomes, Circulating tumor DNA, Circulating tumor cells, Tumor biomarkers, Tumor microenvironment

Introduction

Tumors are heterogeneous diseases that arise from the malignant transformation of somatic cells and can be classified as benign or malignant tumors.1 Early and accurate diagnosis of tumors is crucial for improving treatment efficacy. The diagnosis of tumors primarily relies on pathological examination,2 imaging examination,3 and cytological examination.4 For example, Balaur and colleagues created a microscope slide with an innovative optical design that enables various cells and tissues to display various colors without the need for additional processing. By observing these color variations, researchers successfully distinguished between healthy epithelial cells, precancerous tissue, and breast cancer tissue.5 Yuan et al.6 designed a strategy that relies on intracellular self-assembly controlled by proteinase to concurrently enhance magnetic resonance imaging signals. This approach not only enables the diagnosis and imaging of tumors but also facilitates the real-time monitoring of drug distribution for image-guided tumor therapy. Stephen et al.7 explored fine needle aspiration cytology (FNAC) for diagnosing orbital lymphoid tissue tumors. FNAC, being a minimally invasive and effective method, provides essential cytological diagnostic information for orbital lymphoid tissue tumors, particularly malignant tumors. In addition, recent advances in metal composite sensors,8 including a self-calibrating magnetic aptamer sensor for PD-L1-positive exosomes,9 contribute to innovation in oncological diagnostics. These studies not only broaden the spectrum of tumor diagnostic techniques but also enhance their reliability in clinical applications. Early and accurate tumor diagnosis holds great significance in improving cure rates and survival rates, guiding individualized treatments, predicting patient prognosis, formulating follow-up strategies, conducting high-risk population screening, and avoiding overtreatment. The treatment of smaller tumors and those with slower growth rates, while accurate determination of tumor types and characteristics, allows for the development of appropriate targeted therapy. Currently, the key focus for advancing tumor diagnosis and treatment is enhancing screening sensitivity and specificity, achieving non-invasive and minimally invasive detection, guiding precise treatments, and monitoring treatment responses. The exploration of new diagnostic strategies holds significant value in tumor treatment in the future.

CRISPR/Cas technology, with its programmable target recognition and cleavage capabilities, has demonstrated great potential in tumor biosensing and diagnosis.10,11 This technology refers to the recognition and modification of target genes via various tool enzymes, including in-cell gene editing and somatic cell gene editing techniques.12 Three primary types of gene editing technologies are zinc-finger nucleases (ZFN),13 transcription activator-like effector nucleases (TALEN), and CRISPR/Cas.14 Among them, the CRISPR/Cas system is currently the most widely used and achieves specific cleavage of DNA or RNA through various Cas nucleases.15 The CRISPR/Cas system presents both advantages and challenges in tumor diagnosis. Compared to ZFN and TALENs, CRISPR systems have the advantage of being easier to design and customize, offering greater flexibility in laboratory studies. Additionally, they show greater efficiency in gene editing, which is crucial for accurately targeting diagnostic biomarkers. Despite potential challenges such as off-target effects, these risks can be mitigated through the design of more specific guide RNAs (gRNAs) selected with the aid of advanced algorithms and prediction tools. Compared to traditional diagnostic methods, CRISPR has the advantage of high specificity, allowing for more accurate identification and editing of tumor biomarkers, enabling real-time monitoring for accelerated results. However, it is important to note that CRISPR technology can create technical complexity for researchers with limited experience in gene editing, as well as challenges in delivery systems, especially when applied in vivo. Cas9 cleaves double-stranded DNA near the Protospace Adjacent Motif (PAM) sequence after the hybridization between the single guide RNA (sgRNA) and target DNA.16,17 Cas9 systems, employed for precise gene editing, are strategically adapted for tumor marker detection. Design considerations involve selecting highly specific target sequences to ensure accurate detection of particular tumor markers. The adaptation strategy employs gRNA to guide Cas9, facilitating efficient recognition and precise cutting of target genes. This approach ensures a highly specific response during the detection of tumor markers.18 The mechanism of Cas12 is to initiate dual cleavage after the hybridization between the crRNA and DNA, and the activated Cas12 can cleave along the chain of crRNA and DNA (cis cleavage) as well as nonspecifically cleave the other chain (trans cleavage).19 For the Cas12 system, utilized for specific target gene identification, design considerations involve selecting highly specific target sequences suitable for Cas12 in the context of tumor markers. The adaptation strategy includes designing specific gRNAs to ensure precise cuts on target genes by Cas12. Furthermore, the system exhibits a notable signal amplification effect after cutting the target, contributing to enhanced detection sensitivity. Cas13, employed for RNA detection, recognizes RNA sequences and nonspecifically cleaves nearby RNA guided by designed crRNA.20 For the Cas13 system, widely used in RNA detection, particularly for certain tumor markers involving RNA, design considerations encompass the suitability for RNA detection. The adaptation strategy involves optimizing the gRNA design for Cas13 to ensure high specificity for target RNA. Leveraging the “cleavage activation” mechanism of Cas13 enhances the effective detection of tumor markers. These design considerations and adaptive strategies collectively ensure optimal specificity and sensitivity for each CRISPR/Cas system in detecting diverse tumor markers. Compared to traditional methods, CRISPR/Cas-based biosensing has potential advantages in operational procedures, detection time, and analytical sensitivity. This not only enables precise tumor diagnosis but also predicts tumor occurrence and development, determines prognosis, and guides the selection of treatment plans, thereby greatly improving the level of tumor diagnosis and treatment.21,22

This paper discusses the applications of the CRISPR/Cas system in tumor diagnosis, including the detection of tumor exosomes, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), biomarkers, and tumor microenvironment (TME) factors. A diagram of the study is shown in Figure 1. Firstly, the detection principles and advantages of these applications are introduced. Then, the effects of various CRISPR/Cas-based detection strategies in tumor diagnosis are described. Finally, the prospects of CRISPR/Cas-based biosensing for tumor diagnosis are discussed, aiming to provide references for early tumor detection and precision treatment.

Applications of CRISPR/Cas-based biosensing strategies in tumor diagnosis.
Fig. 1  Applications of CRISPR/Cas-based biosensing strategies in tumor diagnosis.

Detection of exosomes

Exosomes, small vesicles secreted by cells, carry components such as proteins, lipids, mRNAs, and miRNAs. Their surface contains specific protein markers, such as CD9 and CD63. Exosomes play important roles in cell-to-cell signaling, tumor metastasis, immune regulation, and other processes.23,24 CRISPR/Cas-based exosome detection strategies can effectively improve detection sensitivity, expand the range of detectable tumors, and potentially achieve non-invasive early tumor diagnosis and non-invasive “liquid biopsy”. Researchers have achieved specific capture and identification of tumor exosomes by designing CRISPR-Cas systems targeting tumor-related proteins or RNA,25,26 and then introduced various signal amplification strategies to achieve highly sensitive detection.27,28 Xing et al. proposed an innovative approach, apta-HCR-CRISPR assay, for the direct and highly sensitive detection of tumor-derived extracellular vesicle (TEV) protein biomarkers.29 The schematic diagram is illustrated in Figure 2a.29–32 Combining hybridization chain reaction (HCR) and CRISPR-Cas12a, the method achieved a low limit of detection (LOD) of 102 particles/µL, surpassing traditional assays like aptamer-ELISA and apta-HCR-ELISA. Application to clinical samples revealed diagnostic potential for nucleolin+ TEVs in nasopharyngeal carcinoma (NPC) and therapeutic monitoring using PD-L1+ TEVs. The assay’s simplicity suggests its utility for sensitive and versatile TEV protein quantification in clinical settings. Ding et al.30 designed a CRISPR-Cas12a/Cas13a dual system driven by DNAzyme walkers. The schematic representation is shown in Figure 2b. They constructed two DNAzyme walker probes that specifically bind to two tumor-related exosome surface proteins. When probes bind to their targets, DNAzyme cleavage reactions are initiated, releasing DNA sequences that guide gene editing. The activated Cas enzymes then cleave fluorescence labeled short chains, generating a fluorescent signal. This system can sensitively detect exosome proteins in various body fluid samples and achieve intelligent diagnosis through the fluorescent ratio of the two Cas enzymes. The assay is highly sensitive (with limits of detection as low as 30.00 pg/mL for serum amyloid A-1 protein and 200.00 pg/mL for coagulation factor V), highly specific, and ideally accurate. Moreover, this approach enables the simultaneous detection of multiple targets in tumor exosomes and demonstrates the potential of liquid biopsy technology. Yi et al.31 reported a fluorescent aptamer sensor based on magnetic nanoparticles, CRISPR/Cas12a system, and terminal transferase for detecting exosomes from nasopharyngeal carcinoma. The illustrative diagram is presented in Figure 2c. They coupled the Cas12a enzyme with terminal deoxynucleotidyl transferase (TdT), and once the target gene sequence was recognized, the activated Cas12a guided the TdT catalytic reaction to synthesize repetitive sequences and produce a fluorescent signal. Under the optimized conditions, the ability of the fabricated fluorescence aptasensor to detect nasopharyngeal carcinoma-derived exosomes was excellent, with a linear range between 500 to 5 × 104 particles mL−1 and a limit of detection of 100 particles mL−1. Liu et al.32 proposed a method for detecting tumor exosome miR-21 via a gene editing system, magnetic nanoparticles, and cascade displacement reaction fluorescence. Figure 2d illustrates the conceptual diagram. Utilizing the chain displacement activity of the Cas12a enzyme activated by miR-21, they constructed a cascade signal amplification system, using magnetic nanoparticles for exosome extraction and enrichment from the samples. The CRISPR-Cas system can transform exosome detection into specific nucleic acid or protein recognition,33,34 with higher integration and simpler operation,35,36 enabling synchronous detection of multiple components such as miRNAs and proteins.37,38 These methods overcome the challenges of detecting exosomes with extremely low abundance, achieve high-throughput and precise analysis of trace tumor exosomes in body fluids, and broaden the possibilities for accurate diagnosis of exosomes.39

The CRISPR-Cas system combined with multiple signal amplification strategies can detect tumor exosome components.
Fig. 2  The CRISPR-Cas system combined with multiple signal amplification strategies can detect tumor exosome components.

(a) Utilizing HCR and CRISPR-Cas12a double amplification technique, a highly sensitive apta-HCR-CRISPR method was devised for the detection of TEV protein.29 (b) A novel CRISPR-Cas12a/Cas13a approach, activated by DNAzyme walkers, is suggested for the concurrent identification of exosomal proteins such as SAA1 and clotting FV.30 (c) A fluorescence aptasensor was created to detect NPC-derived exosomes by utilizing a combination of MNPs, TdT, and CRISPR/Cas12a.31 (d) An innovative biosensor, incorporating MNPs, CSDR, and CRISPR/Cas12a, was created to identify the exosome miR-21 in lung cancer.32 Cas12a, CRISPR-associated protein 12a; Cas13a, CRISPR-associated protein 13a; CD, Cluster of Differentiation; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; crRNA, CRISPR RNA; DL, Deep Learning; EVs, Extracellular Vesicles; FQ, fluorophore–quencher reporter;PD-L1, Programmed Death-Ligand 1. (a) Reproduced under the applicable open-access license. (b) Reproduced with permission from Elsevier. (c) Reproduced with permission from Springer Nature. (d) Reproduced under the applicable open-access license.

Circulating tumor DNA testing

ctDNA refers to fragmented DNA released by tumor cells undergoing apoptosis or necrosis, which can be obtained from blood samples.40 It represents the entire tumor genome and can be used as a real-time and dynamic monitor for tumor changes. It serves as a crucial indicator for liquid biopsy of tumors, although its concentration in samples is relatively low.41 Chen et al. utilized a complex of Cas9 protein and sgRNA to target and cleave ctDNA,42 exposing hydroxyl groups captured by 3D graphene/AuPtPd nanoflower materials. As depicted in Figure 3a,42–44 the diagram provides a visual representation. Subsequently, platinum ions are released and deposited on the working electrode surface, generating a strong electrochemical signal. With these methods, the content of ctDNA in the sample can be further calculated. Zhou et al.43 devised a cascaded amplification system employing CRISPR/Cas12a. The schematic representation is shown in Figure 3b. Upon recognition of the target DNA by the first-level Cas12a, the second-level Cas12a is triggered to amplify the signal. Spherical nucleic acids serve as reporting molecules due to their high stability and exhibit a notable signal amplification effect, leading to enhanced stability and sensitivity (5 orders of magnitude). Li et al.44 proposed a targeted amplification strategy by designing gRNAs that specifically target ctDNA sequences, identifying and cleaving them to achieve highly selective amplification. The diagram in Figure 3c serves as a visual representation. Subsequently, a protonated probe is introduced, which hybridizes with the adjacent cut end of the ctDNA, triggering a fluorescent signal and achieving dual signal amplification of the product, thus enabling precise detection of ctDNA with extremely low abundance in body fluid samples. This strategy exhibits high sensitivity for ctDNA detection with a low limit of 5.43 fM. Liu et al.45 employed an electrochemical method to detect the EGFR gene in ctDNA from non-small cell lung cancer (NSCLC) patients. They designed crRNAs targeting the EGFR gene’s 19del and L858R mutation sites, which were mixed with Cas12a, substrate strands, and the target ctDNA. When the crRNA recognizes the mutation site, Cas12a cleaves the substrate strand, generating an electrochemical signal. Wang et al.46 also proposed a method for detecting mutations in ctDNA from NSCLC patients. They designed a crRNA library targeting various gene mutation sites, such as those in EGFR, KRAS, and TP53. The ctDNA was pre-amplified using multiplex PCR to enrich the mutation sites, and then Cas12a was employed with the crRNA library. When the crRNA paired with the target mutation, Cas12a was activated, and the probes were cleaved, resulting in a fluorescent signal. This method achieved high-throughput, multi-site parallel detection of common driver gene mutations in NSCLC, offering broader applicability compared to single-target detection. Wu et al.47 proposed a chain displacement amplification fluorescence resonance energy transfer technique. This technique utilized the non-specific cleavage activity of the Cas12a enzyme and introduced initiation probes and reporting probes to initiate a chain displacement reaction and amplify the signal.48 These studies have designed targeted CRISPR/Cas-based recognition and cleavage models, combined with signal amplification and techniques such as electrochemistry and fluorescence.49 Extremely low abundance ctDNA and mutations in bodily fluid samples have been successfully detected.50 This approach has the potential to greatly improve the accuracy of early cancer diagnosis.51

The CRISPR-based recognition and cleavage model, in conjunction with signal amplification and detection techniques such as electrochemistry and fluorescence, was able to identify ctDNA.
Fig. 3  The CRISPR-based recognition and cleavage model, in conjunction with signal amplification and detection techniques such as electrochemistry and fluorescence, was able to identify ctDNA.

(a) A novel 3D GR/AuPtPd nanoflower sensing platform, based on CRISPR/Cas9 cleavage-triggered ESDR, has been developed for the effective detection of ctDNA.42 (b) An amplifier utilizing spherical cascade CRISPR/Cas12a and nucleic acid reporter was designed to enhance the accuracy and responsiveness of ctDNA detection.43 (c) A method was devised for the precise identification of ctDNA through the utilization of HCR and adjacent hybridization regulated CRISPR/Cas12a, employing a dual-signal amplification strategy.44 ctDNA, circulating tumor DNA; DPV, differential pulse voltammetr; ESDR, entropy-driven strand displacement reaction; GCE, glassy carbon electrode; GR, graphene; HCR, hybridization chain reaction; HDPC, high definition photoconductor. (a) Reproduced with permission from Elsevier; (b) Reproduced with permission from Royal Society of Chemistry. (c) Reproduced with permission from Elsevier.

Detection of CTCs

CTCs are tumor cells that detach from primary tumors or metastatic lesions and enter the circulation.52 They play a crucial role in tumor progression and metastasis.53 Various biosensing strategies have been developed for the detection and analysis of CTCs.54,55 CRISPR/Cas-based approaches have further shown potential for sensitive CTC detection.56,57 Lv et al.56 designed and constructed a multivalent double-stranded DNA aptamer network that specifically recognizes tumor surface markers. Figure 4a displays the conceptual diagram.56,58,59 Upon binding the aptamer to the target, the connected Cas12a enzyme is activated to cleave the reporter gene, generating a fluorescent signal. The detection limit of this method can reach 26 cells/mL. Wang et al.57 designed a dual-nucleic acid adapter DNA network to enhance the specific capture of CTCs. Using CRISPR/Cas12a as a signal amplifier, the DNA network can release captured CTCs for subsequent analysis through chemotaxis by inputting a release signal DNA strand. The specificity and sensitivity of the test were demonstrated in human blood samples. Yin et al.58 developed a fluidly confined CRISPR-based DNA reporter (FINDER) on live cell membranes for the rapid and sensitive identification of cancer cells. The schematic illustration is shown in Figure 4b. FINDER consists of sgRNA and Cas proteins that target specific sequences of cancer-associated genes. When FINDER interacts with the target sequences, it triggers the enzymatic activity of Cas proteins, releasing a fluorescent signal for real-time detection of live cells. The FINDER rapidly identified target cells in only 20 min, and achieved over 80% recognition efficiency even with only 0.1% of the target cells being present in clinical blood samples. This approach holds great potential for precision medicine and biosensing applications. Ren et al.59 designed a histone-composed nanodelivery system for in situ detection of biomarkers at the single-cell level. The schematic representation is presented in Figure 4c. They combined histones with CRISPR/Cas9 plasmids and surface-modified hyaluronic acid to target CD44, as well as a fusion peptide of T22 sequence to target CXCR4 and a nuclear localization sequence. This system selectively delivered the gene editing plasmids to CTCs in whole blood, especially those overexpressing CD44 and CXCR4, enabling efficient operation of gene editing. Molecular beacons for p53 and p21 were loaded and detected within the delivery system at a single-cell level. In addition, gene editing technology not only can be used to repair disease-causing genes for treatment,60 but also to detect the expression and mutations of specific genes, evaluate the risk of drug resistance in tumors, and guide individualized drug treatment,61 laying the theoretical foundation for precision medicine.62

An innovative CTC detection system, utilizing DNA aptamers and CRISPR-Cas effectors, was devised to attain precise capture and precise detection of CTC.
Fig. 4  An innovative CTC detection system, utilizing DNA aptamers and CRISPR-Cas effectors, was devised to attain precise capture and precise detection of CTC.

(a) A sensor that detects CTCs using CRISPR-Cas12 MDANs has been reported.56 (b) A FINDER was successfully developed on living cell membranes, enabling the rapid and sensitive identification of cancer cells in clinical blood samples.58 (c) The reprogramming of CTCs with in situ detection of biomarkers at the single-cell level is achieved through the utilization of multi-functionalized nano-systems containing a delivery vector consisting of histone for plasmid loading, hyaluronic acid for tumor targeting, and a fusion peptide for CXCR4 targeting, along with nuclear localization.59 CTC, circulating tumor cell; MDANs, multivalent duplexed-aptamer networks; FINDER, fluidly confined CRISPR-based DNA reporter; CXCR4, C-X-C motif chemokine receptor 4; MMP, Matrix Metalloproteinase. (a) Reproduced under the applicable open-access license. (b) Reproduced with permission from Wiley. (c) Reproduced under the applicable open-access license.

Detection of tumor biomarkers

Tumor biomarkers are a series of biological indicators associated with the formation and progression of tumors, including molecular markers reflecting genetic changes, related cytokines and receptors, tumor cell-specific metabolic products, enzymes with increased activity or expression, and antigens suggesting the occurrence of tumors.63 These biomarkers serve multiple purposes, such as tumor detection, classification, prognosis assessment, and clinical observation.63,66 Combining CRISPR/Cas-based biosensing with immunodetection, DNA nanotechnology, and various signal amplification techniques can enable highly sensitive detection of key tumor biomarkers.64,65,67 For example, telomerase activity is suppressed in normal human tissues, but is reactivated in approximately 90% of cancer cells, allowing them to maintain telomere length during cell division and achieve immortality.68 Therefore, aberrant telomerase activity is considered to be associated with various tumors.69 Chen et al.70 designed a special double-stranded DNA structure as a substrate, in which the guiding sequence and the reporting sequence bind complementarily to form a helical structure. Figure 5a shows a depiction of the conceptual diagram.70,73–75 When telomerase cleaves the guiding sequence, the DNA structure is unwound, releasing the guiding sequence to bind to Cas12a, activating Cas12a’s cleavage activity to cut the fluorophore on reporting sequence, generating a fluorescent signal. Prostate-specific antigen (PSA) is secreted by prostate epithelial cells and belongs to the kallikrein family of proteins. It is present in prostate tissue and semen, and its concentration in normal human serum is extremely low. PSA is the preferred biomarker for diagnosing prostate cancer. Liang et al.71 linked anti-PSA antibodies to Cas12a. When PSA is present, it binds to the antibody, inhibiting the endonuclease activity of Cas12a, thereby suppressing the cleavage of the reporting molecule, resulting in a decreased fluorescent signal. The optical density transducer created by platinum nanoparticles (OD-CRISPR) assay achieved a minimal detectable concentration of 0.01 ng/mL. Jia et al.72 utilized the CRISPR system along with a personal blood glucose meter for the quantitative detection of the liver cancer biomarker alpha-fetoprotein (AFP). The detection module comprises a gRNA to guide the CRISPR system, Cas12a enzyme linked to glucose oxidase, and a reporting module. They quantitatively detected the AFP biomarker in spiked human serum samples with a detection sensitivity down to 10 ng/mL. In the presence of AFP, it specifically binds to the gRNA, activating’s enzyme activity of Cas12a. This, in turn, cleaves the sequence linking glucose oxidase, releasing the enzyme to catalyze glucose and generate glucuronic acid, which can be detected using a personal blood glucose meter. In another innovative approach, Hong et al.39 combined the CRISPR/Cas13a system with liposome technology for direct detection of trace amounts of miRNA in cellular and bodily fluid samples without nucleic acid extraction and amplification. They detected miR-21-5p in plasma samples from ovarian cancer patients. Additionally, Yang et al.73 designed a CRISPR-Cas12a-based protease-inducible transcription activation system, where the protease cleaves the peptide chain to activate downstream gene expression and drive the CRISPR-Cas12a system, enabling highly sensitive detection of tumor-associated protease biomarkers. Figure 5b provides an illustration of the concept. In the subsequent study, they overcame the limitation of CRISPR-Cas system in detecting only nucleic acid by designing DNA linker reactions, enabling the detection of tumor-associated proteins and small molecules.74 The diagram presented in Figure 5c provides a visual representation. Chen et al.75 combined CRISPR/Cas13a with immunoassay technology to design a dual signal amplification mechanism, greatly improving the detection sensitivity of biomarker proteins. The conceptual diagram is visually represented in Figure 5d. Liu et al.76 integrated the CRISPR/Cas13a system with gold nanoparticles technology, achieving direct detection of low-abundance RNA in cells without nucleic acid amplification or labeling. Zhao et al.77 proposed a multifunctional biosensing platform integrating the CRISPR-Cas12a system with apatamers, which can detect a variety of target molecules. Chen et al.78 integrated CRISPR-Cas12a with rolling circle amplification, significantly improving the detection sensitivity of circular non-coding RNA. Wu et al.79 used the CRISPR/Cas13a system with transcription-mediated signal amplification to achieve highly sensitive detection of the important tumor immunotherapy response marker IL-15 mRNA. These studies demonstrate the potential of CRISPR/Cas-based biosensing in detecting nucleic acid and non-nucleic acid biomarkers,80 greatly expanding the detection applications of gene editing technology and promoting rapid development in this field.81,82 With the gradual improvement in detection sensitivity, it is foreseeable that CRISPR-Cas technology has the potential to achieve precise diagnosis and prediction of tumors at an early stage.83,84

The innovative combination of CRISPR/Cas technology with immunoassay, DNA nanotechnology, and multiple signal amplification technologies provides a fundamental platform for tumor marker monitoring.
Fig. 5  The innovative combination of CRISPR/Cas technology with immunoassay, DNA nanotechnology, and multiple signal amplification technologies provides a fundamental platform for tumor marker monitoring.

(a) A CRISPR-Cas12a system was designed that uses a generalized stimulus-response switching mechanism based on pcDNAs. DNA substrates that do not necessitate the PAM and bubble structure are used to construct stimulation-responsive pcDNAs. The CRISPR-Cas12a transcleavage capability was then employed for signal reporting, and a multifunctional CRISPR-based living cell biosensing system was set up to identify tumor markers.70 (b) The utilization of both protease-induced transcriptional activation and CRISPR-Cas12a technology allowed for the identification of tumor marker protease.73 (c) The purpose of this paper is to elucidate a CRISPR-Cas12a sensing platform that is activated by DNA linking reactions triggered by biomolecules, leading to the production of DNA double strands that activate Cas12a’s nuclease activity. Consequently, this platform generates an amplified fluorescent signal, enabling the sensitive detection of non-nucleic acid targets, including NAD+. ATP and PNK are involved in the process.74 (d) The mechanism utilizes a CRISPR/ Cas13-based signal output amplification strategy to double the output signal through T7 RNA polymerase transcription and CRISPR/Cas13a side cutting activity. Detection of inflammatory factor human IL-6 and tumor marker human VEGF.75 NUC, nuclease lobe; REC, recognition lobe; pcDNAs, PAM-less conditional DNA substrates; PAM, protospacer-adjacent motif; PNK, polynucleotide kinase; VEGF, vascular endothelial growth factor. (a) Reproduced with permission from Royal Society of Chemistry. (b) Reproduced with permission from Springer Nature. (c) Reproduced with permission from Royal Society of Chemistry. (d) Reproduced with permission from American Chemical Society.

Related CRISPR applications in TME analysis

TME is the intra- and extra-cellular microenvironment surrounding tumor tissues, including various stromal cells, immune cells, and the vascular system coexisting with tumor cells, as well as non-cellular components such as extracellular matrix, cytokines, and chemotactic factors.85 The TME has complex interactions with tumor cells and provides a suitable soil for tumor initiation, development, invasion, and metastasis, playing a crucial role in the progression of tumors.86 Dhainaut et al.87 developed a gene knockout screening system on tissue slices to preserve tissue structure and microenvironmental information. By conducting gene knockouts in human and mouse tumor samples, they used image analysis to evaluate changes in the microenvironment and identified several potential regulators of the TME. This study demonstrated the potential of combining CRISPR gene editing technology with histological analysis for detecting the TME. Dervovic et al.88 used in vivo CRISPR gene knockout screening to identify genes Serpinb9 and Adam2 that are involved in regulating the immune response to lung cancer immunotherapy. In addition, there are studies that utilize gene editing technology to modify the TME, including targeting T cells, CAR-T cells,89 tumor-associated macrophages,90 etc., to enhance the effectiveness of tumor immunotherapy or identify key genes regulating tumor immunotherapy.91 These efforts provide important insights and tools for the development of more effective precise immunotherapies.

Prospects

CRISPR/Cas-based biosensing holds great potential in advancing tumor diagnosis. By combining programmable target recognition with different signal amplification strategies, CRISPR/Cas systems can facilitate sensitive detection of tumor-associated nucleic acids, proteins, and other biomarkers in blood or other biological samples. Advantages of CRISPR/Cas system in detecting exosomes, ctDNA, CTCs, and tumor markers are summarized in Table 1.30,31,43,44,56,58,71,72 However, there are still several bottlenecks remain before clinical translation. Firstly, molecular diagnosis is not equal to clinical diagnosis currently. False-positive and false-negative results can lead to misdiagnosis, and clinical diagnosis requires other methods for synthetic judgments. Secondly, there is a need to improve the specificity and sensitivity of cancer biomarkers in the CRISPR/Cas system. Finally, the ethical considerations should not be overlooked. One of the ethical challenges and mitigation strategies is privacy protection: the use of CRISPR technology may involve obtaining and modifying personal genetic information. To protect patient privacy, researchers should comply with relevant ethical guidelines and regulations, using measures such as anonymization and encryption. The second is patient informed consent: Patients should fully understand the potential risks and benefits of CRISPR technology. Transparent communication and comprehensive patient education are key to addressing ethical issues, and informed consent should be obtained before any gene-editing procedure is undertaken. These issues should be addressed to support the responsible clinical translation of CRISPR/Cas-based biosensing.

Table 1

Advantages of CRISPR/Cas system in detecting exosomes, ctDNA, CTCs, and tumor markers

TitleMethodObjectsDetection capabilityRef
Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in the intelligent diagnosis of cancerCas12a/Cas13aexosomelimits of detection as low as 30.00 pg/mL for serum amyloid A-1 protein and 200.00 pg/mL for coagulation factor V30
Fluorescent aptasensor based on the MNPs-CRISPR/Cas12a-TdT for the determination of nasopharyngeal carcinoma-derived exosomes.Cas12aexosomelinear range between 500 to 5 × 104 particles mL−1 and the limit of detection of 100 particles mL−131
Spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier for stable and sensitive biosensing of circulating tumor DNACas12actDNAImproved stability and sensitivity (5 orders of magnitude)43
Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNACas12actDNAdetection limit of 5.43 fM44
Multivalent Duplexed-Aptamer Networks Regulated a CRISPR Cas12a System for CTC DetectionCas12aCTCsThe detection limit is 26 cells/ml56
FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell IdentificationCRISPRCTCsThe FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples58
Platinum nanoparticles-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samplesCas12atumor biomarkersThe lowest detection concentration was 0.01 ng/mL71
CRISPR-powered biosensing platform for quantitative detection of alpha-fetoprotein by a personal glucose meterCRISPRtumor biomarkersThe detection sensitivity is as low as 10 ng/mL72

Cas12a/Cas13a, CRISPR-associated protein 12a/CRISPR-associated protein 13a; ctDNA, circulating tumor DNA; CTCs, circulating tumor cells; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; FINDER, a fluidly confined CRISPR-based DNA reporter; MNPs, magnetic nanoparticles.

Limitations

This narrative review has several limitations. The literature was not identified using a predefined systematic search or formal quality-assessment process; therefore, some selection bias may be present. In addition, the included studies used heterogeneous targets, sample types, assay designs, and performance measures, which limits direct comparison among different CRISPR/Cas-based biosensing strategies.

Conclusions

CRISPR/Cas-based biosensing strategies are rapidly developing for the detection of tumor-derived exosomes, ctDNA, CTCs, and tumor biomarkers. Current studies demonstrate promising analytical sensitivity and flexible integration with different signal amplification platforms; however, further standardization and clinical validation are needed before these approaches can be routinely applied to cancer screening or diagnosis. Continued development and validation may facilitate the translation of CRISPR/Cas-based biosensing toward precision cancer diagnosis.

Abbreviations

ctDNA: 

circulating tumor DNA

CTCs: 

circulating tumor cells

FNAC: 

fine needle aspiration cytology

ZFN: 

zinc-finger nucleases

TALEN: 

transcription activator-like effector nucleases

PAM: 

protospace adjacent motif

sgRNA: 

single guide RNA

gRNAs: 

guide RNAs

SDA: 

strand displacement amplification

AFP: 

alpha-fetoprotein

TME: 

tumor microenvironment

HCR: 

hybridization chain reaction

MNPs: 

magnetic nanoparticles

AFP: 

alpha-fetoprotein

Cas12a: 

CRISPR-associated protein 12a

Cas13a: 

CRISPR-associated protein 13a

CD: 

cluster of differentiation

CRISPR: 

clustered regularly interspaced short palindromic repeats

crRNA: 

CRISPR RNA

CTCs: 

circulating tumor cells

ctDNA: 

circulating tumor DNA

DL: 

deep learning

DPV: 

differential pulse voltammetry

dTTP: 

deoxythymidine triphosphate

EVs: 

extracellular vesicles

FNAC: 

fine needle aspiration cytology

FQ: 

fluorophore–quencher reporter

GCE: 

glassy carbon electrode

GR: 

graphene

gRNAs: 

guide RNAs

HCR: 

hybridization chain reaction

HDPC: 

high definition photoconductor

MMP: 

matrix metalloproteinase

MNPs: 

magnetic nanoparticles

NLS: 

nuclear localization signal

NUC: 

nuclease lobe

PAM: 

protospace adjacent motif

PD-L1: 

programmed death-ligand 1

REC: 

recognition lobe

SDA: 

strand displacement amplification

sgRNA: 

single guide RNA

TALEN: 

transcription activator-like effector nucleases

TME: 

tumor microenvironment

ZFN: 

zinc-finger nucleases

Declarations

Acknowledgement

None.

Funding

This work was supported by the Joint Funds for the Innovation of Science and Technology, Fujian province (Grant number: 2021Y9014) and the Fujian Medical University’s Research Foundation for Talented Scholars (No. XRCZX2019024).

Conflict of interest

The authors have no conflict of interests related to this publication.

Authors’ contributions

Literature review and manuscript writing (QLY and XYZ), drafting figures (LMZ and HZ), and topic conception and critical revision (SWH). All authors have made significant contributions to this study and approved the final manuscript.

References

  1. Fu Y, Jung AW, Torne RV, Gonzalez S, Vöhringer H, Shmatko A, et al. Pan-cancer computational histopathology reveals mutations, tumor composition and prognosis. Nat Cancer 2020;1(8):800–810 View Article PubMed/NCBI
  2. Tsai PC, Lee TH, Kuo KC, Su FY, Lee TM, Marostica E, et al. Histopathology images predict multi-omics aberrations and prognoses in colorectal cancer patients. Nat Commun 2023;14(1):2102 View Article PubMed/NCBI
  3. Thakur NA, Daniels AH, Schiller J, Valdes MA, Czerwein JK, Schiller A, et al. Benign tumors of the spine. J Am Acad Orthop Surg 2012;20(11):715–724 View Article PubMed/NCBI
  4. Kennedy GT, Azari FS, Bernstein E, Nadeem B, Chang A, Segil A, et al. Targeted detection of cancer at the cellular level during biopsy by near-infrared confocal laser endomicroscopy. Nat Commun 2022;13(1):2711 View Article PubMed/NCBI
  5. Balaur E, O’Toole S, Spurling AJ, Mann GB, Yeo B, Harvey K, et al. Colorimetric histology using plasmonically active microscope slides. Nature 2021;598(7879):65–71 View Article PubMed/NCBI
  6. Yuan Y, Zhang J, Qi X, Li S, Liu G, Siddhanta S, et al. Furin-mediated intracellular self-assembly of olsalazine nanoparticles for enhanced magnetic resonance imaging and tumour therapy. Nat Mater 2019;18(12):1376–1383 View Article PubMed/NCBI
  7. Stephen N, Manivannan P, Gochhait D, Sreerekha J, Ramasubramanian N, Srinivas BH, et al. The utility of fine needle aspiration cytology in orbital haematolymphoid neoplasms. Cytopathology 2021;32(2):217–226 View Article PubMed/NCBI
  8. Zhang Y, Liu X, Li N, Xu Y, Ma Y, Huang Z, et al. Typing of cancer cells by microswimmer based on Co-Fe-MOF for one-step simultaneously detect multiple biomarkers. Biosens Bioelectron 2023;230:115263 View Article PubMed/NCBI
  9. Zhu N, Wang X, Zhang Y, Mao Y, Yu Y, Yi Q, et al. Self-calibrated magnetic aptamer sensor with dual Lanthanide-assisted Time-resolved luminescence for high-sensitive detection of melanoma exosomal PD-L1. Mater Design 2023;227:111714 View Article PubMed/NCBI
  10. Hao L, Zhao RT, Welch NL, Tan EKW, Zhong Q, Harzallah NS, et al. CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics. Nat Nanotechnol 2023;18(7):798–807 View Article PubMed/NCBI
  11. Wang M, Chen M, Wu X, Huang X, Yu B. CRISPR applications in cancer diagnosis and treatment. Cell Mol Biol Lett 2023;28(1):73 View Article PubMed/NCBI
  12. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816–821 View Article PubMed/NCBI
  13. Paschon DE, Lussier S, Wangzor T, Xia DF, Li PW, Hinkley SJ, et al. Diversifying the structure of zinc finger nucleases for high-precision genome editing. Nat Commun 2019;10(1):1133 View Article PubMed/NCBI
  14. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819–823 View Article PubMed/NCBI
  15. Wang JY, Doudna JA. CRISPR technology: A decade of genome editing is only the beginning. Science 2023;379(6629):eadd8643 View Article PubMed/NCBI
  16. Yang M, Sun R, Deng P, Yang Y, Wang W, Liu JG, et al. Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search. Chem Sci 2021;12(38):12776–12784 View Article PubMed/NCBI
  17. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 2014;343(6176):1247997 View Article PubMed/NCBI
  18. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014;156(5):935–949 View Article PubMed/NCBI
  19. Li SY, Cheng QX, Liu JK, Nie XQ, Zhao GP, Wang J. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res 2018;28(4):491–493 View Article PubMed/NCBI
  20. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, et al. RNA editing with CRISPR-Cas13. Science 2017;358(6366):1019–1027 View Article PubMed/NCBI
  21. Durán-Vinet B, Araya-Castro K, Calderón J, Vergara L, Weber H, Retamales J, et al. CRISPR/Cas13-Based Platforms for a Potential Next-Generation Diagnosis of Colorectal Cancer through Exosomes Micro-RNA Detection: A Review. Cancers (Basel) 2021;13(18):4640 View Article PubMed/NCBI
  22. Zhang J, You Y. CRISPR-Cas13a system: a novel approach to precision oncology. Cancer Biol Med 2020;17(1):6–8 View Article PubMed/NCBI
  23. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020;367(6478):eaau6977 View Article PubMed/NCBI
  24. Yu D, Li Y, Wang M, Gu J, Xu W, Cai H, et al. Exosomes as a new frontier of cancer liquid biopsy. Mol Cancer 2022;21(1):56 View Article PubMed/NCBI
  25. Ye Y, Shi Q, Yang T, Xie F, Zhang X, Xu B, et al. In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System. Technol Cancer Res Treat 2022;21:15330338221085370 View Article PubMed/NCBI
  26. Li J, Xie S, Qu F, Tan W. Aptasensors for Cancerous Exosome Detection. Methods Mol Biol 2022;2504:3–20 View Article PubMed/NCBI
  27. Zhao X, Zeng L, Mei Q, Luo Y. Allosteric Probe-Initiated Wash-Free Method for Sensitive Extracellular Vesicle Detection through Dual Cycle-Assisted CRISPR-Cas12a. ACS Sens 2020;5(7):2239–2246 View Article PubMed/NCBI
  28. Zhao X, Zhang W, Qiu X, Mei Q, Luo Y, Fu W. Rapid and sensitive exosome detection with CRISPR/Cas12a. Anal Bioanal Chem 2020;412(3):601–609 View Article PubMed/NCBI
  29. Xing S, Lu Z, Huang Q, Li H, Wang Y, Lai Y, et al. An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification. Theranostics 2020;10(22):10262–10273 View Article PubMed/NCBI
  30. Ding L, Wu Y, Liu LE, He L, Yu S, Effah CY, et al. Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer. Biosens Bioelectron 2023;219:114827 View Article PubMed/NCBI
  31. Yi P, Luo D, Gao Z, Chen Q, Zhou Y. Fluorescent aptasensor based on the MNPs-CRISPR/Cas12a-TdT for the determination of nasopharyngeal carcinoma-derived exosomes. Mikrochim Acta 2023;190(2):74 View Article PubMed/NCBI
  32. Liu Q, Liu J, He N, Zhang M, Wu L, Chen X, et al. CRISPR/Cas12a Coupling with Magnetic Nanoparticles and Cascaded Strand Displacement Reaction for Ultrasensitive Fluorescence Determination of Exosomal miR-21. Molecules 2022;27(16):5338 View Article PubMed/NCBI
  33. Luo SH, Wu Y, Pan WL, Zhong GZ, Situ B, Li B, et al. An integrated magneto-fluorescent nanosensor for rapid and sensitive detection of tumor-derived exosomes. Sens Actuators B 2023;374:132792 View Article PubMed/NCBI
  34. Li CY, Liu JX, Liu YH, Gao JL, Zheng B, Liu D. An exceptional and universal DNA walker amplified “one-to-many” CRISPR/Cas12a-mediated fluorescent biosensor for ultrasensitive detection of non-DNA biomarkers. Sens Actuators B Chem 2022;361:31743 View Article PubMed/NCBI
  35. Shen B, Li L, Liu C, Li X, Li X, Cheng X, et al. Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA. Anal Chem 2023;95(9):4486–4495 View Article PubMed/NCBI
  36. Zhang J, Guan M, Ma C, Liu Y, Lv M, Zhang Z, et al. Highly Effective Detection of Exosomal miRNAs in Plasma Using Liposome-Mediated Transfection CRISPR/Cas13a. ACS Sens 2023;8(2):565–575 View Article PubMed/NCBI
  37. Li H, Xing S, Xu J, He Y, Lai Y, Wang Y, et al. Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins. Talanta 2021;221:121670 View Article PubMed/NCBI
  38. Zhao L, Wang H, Fu J, Wu X, Liang XY, Liu XY, et al. Microfluidic-based exosome isolation and highly sensitive aptamer exosome membrane protein detection for lung cancer diagnosis. Biosens Bioelectron 2022;214:114487 View Article PubMed/NCBI
  39. Hong JS, Son T, Castro CM, Im H. CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles. Adv Sci (Weinh) 2023;10(24):e2301766 View Article PubMed/NCBI
  40. Li X, Ye M, Zhang W, Tan D, Jaffrezic-Renault N, Yang X, et al. Liquid biopsy of circulating tumor DNA and biosensor applications. Biosens Bioelectron 2019;126:596–607 View Article PubMed/NCBI
  41. Melani C, Wilson WH, Roschewski M. Liquid biopsy in non-Hodgkin’s lymphoma. Hematol Oncol 2019;37(Suppl 1):70–74 View Article PubMed/NCBI
  42. Chen M, Wu D, Tu S, Yang C, Chen D, Xu Y. CRISPR/Cas9 cleavage triggered ESDR for circulating tumor DNA detection based on a 3D graphene/AuPtPd nanoflower biosensor. Biosens Bioelectron 2020;173:112821 View Article PubMed/NCBI
  43. Zhou M, Yin Y, Shi Y, Huang Z, Shi Y, Chen M, et al. Spherical nucleic acid reporter-based cascade CRISPR/Cas12a amplifier for stable and sensitive biosensing of circulating tumor DNA. Chem Commun (Camb) 2022;58(28):4508–4511 View Article PubMed/NCBI
  44. Li M, Luo N, Liao X, Zou L. Proximity hybridization-regulated CRISPR/Cas12a-based dual signal amplification strategy for sensitive detection of circulating tumor DNA. Talanta 2023;257:124395 View Article PubMed/NCBI
  45. Liu F, Peng J, Lei YM, Liu RS, Jin L, Liang H, et al. Electrochemical detection of ctDNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system. Sens Actuators B Chem 2022;362:131807 View Article PubMed/NCBI
  46. Wang XL, Song J, Fan XY, Shi CL, Zeng BJ, Xiao YQ, et al. Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system. Sens Actuators B Chem 2023;383:133493 View Article PubMed/NCBI
  47. Wu X, Ju T, Li Z, Li J, Zhai X, Han K. Target-independent hybridization chain reaction-fluorescence resonance energy transfer for sensitive assay of ctDNA based on Cas12a. Anal Chim Acta 2023;1261:341170 View Article PubMed/NCBI
  48. Tsou JH, Leng Q, Jiang F. A CRISPR Test for Rapidly and Sensitively Detecting Circulating EGFR Mutations. Diagnostics (Basel) 2020;10(2):114 View Article PubMed/NCBI
  49. Choi JH, Lim J, Shin M, Paek SH, Choi JW. CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis. Nano Lett 2021;21(1):693–699 View Article PubMed/NCBI
  50. Kim B, Kim Y, Shin S, Lee ST, Cho JY, Lee KA. Application of CRISPR/Cas9-based mutant enrichment technique to improve the clinical sensitivity of plasma EGFR testing in patients with non-small cell lung cancer. Cancer Cell Int 2022;22(1):82 View Article PubMed/NCBI
  51. Zhang W, Zhao S, Xie Z, Chen S, Huang Y, Zhao Z, et al. The fluorescence amplification strategy based on 3D DNA walker and CRISPR/Cas12a for the rapid detection of BRAF V600E. Anal Sci 2022;38(8):1057–1066 View Article PubMed/NCBI
  52. Lin D, Shen L, Luo M, Zhang K, Li J, Yang Q, et al. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther 2021;6(1):404 View Article PubMed/NCBI
  53. Castro-Giner F, Aceto N. Tracking cancer progression: from circulating tumor cells to metastasis. Genome Med 2020;12(1):31 View Article PubMed/NCBI
  54. Shen H, Deng W, He Y, Li X, Song J, Liu R, et al. Ultrasensitive aptasensor for isolation and detection of circulating tumor cells based on CeO2@Ir nanorods and DNA walker. Biosens Bioelectron 2020;168:112516 View Article PubMed/NCBI
  55. Chen Y, Chen X, Li M, Fan P, Wang B, Zhao S, et al. A new analytical platform for potential point-of-care testing of circulating tumor cells. Biosens Bioelectron 2021;171:112718 View Article PubMed/NCBI
  56. Lv Z, Wang Q, Yang M. Multivalent Duplexed-Aptamer Networks Regulated a CRISPR-Cas12a System for Circulating Tumor Cell Detection. Anal Chem 2021;93(38):12921–12929 View Article PubMed/NCBI
  57. Wang DX, Wang J, Wang YX, Ma JY, Liu B, Tang AN, et al. A CRISPR/Cas12a-responsive dual-aptamer DNA network for specific capture and controllable release of circulating tumor cells. Chem Sci 2022;13(35):10395–10405 View Article PubMed/NCBI
  58. Yin Y, Xie W, Xiong M, Gao Y, Liu Q, Han D, et al. FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification. Angew Chem Int Ed Engl 2023;62(44):e202309837 View Article PubMed/NCBI
  59. Ren XH, He XY, Xu C, Han D, Cheng SX. Functional Tumor Targeting Nano-Systems for Reprogramming Circulating Tumor Cells with In Situ Evaluation on Therapeutic Efficiency at the Single-Cell Level. Adv Sci (Weinh) 2022;9(21):e2105806 View Article PubMed/NCBI
  60. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 2014;32(6):551–553. Erratum in: Nat Biotechnol 2014;32(9):952 View Article PubMed/NCBI
  61. Gonzalez-Salinas F, Rojo R, Martinez-Amador C, Herrera-Gamboa J, Trevino V. Transcriptomic and cellular analyses of CRISPR/Cas9-mediated edition of FASN show inhibition of aggressive characteristics in breast cancer cells. Biochem Biophys Res Commun 2020;529(2):321–327 View Article PubMed/NCBI
  62. Lee J, Choi A, Cho SY, Jun Y, Na D, Lee A, et al. Genome-scale CRISPR screening identifies cell cycle and protein ubiquitination processes as druggable targets for erlotinib-resistant lung cancer. Mol Oncol 2021;15(2):487–502 View Article PubMed/NCBI
  63. Filella X, Rodríguez-Garcia M, Fernández-Galán E. Clinical usefulness of circulating tumor markers. Clin Chem Lab Med 2023;61(5):895–905 View Article PubMed/NCBI
  64. Zhou T, Huang R, Huang M, Shen J, Shan Y, Xing D. CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection. Adv Sci (Weinh) 2020;7(13):1903661 View Article PubMed/NCBI
  65. Zhao D, Tang J, Tan Q, Xie X, Zhao X, Xing D. CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection. Talanta 2023;260:124582 View Article PubMed/NCBI
  66. Shimada H, Noie T, Ohashi M, Oba K, Takahashi Y. Clinical significance of serum tumor markers for gastric cancer: a systematic review of literature by the Task Force of the Japanese Gastric Cancer Association. Gastric Cancer 2014;17(1):26–33 View Article PubMed/NCBI
  67. Jia HY, Zhao HL, Wang T, Chen PR, Yin BC, Ye BC. A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction. Biosens Bioelectron 2022;211:114382 View Article PubMed/NCBI
  68. Salamati A, Majidinia M, Asemi Z, Sadeghpour A, Oskoii MA, Shanebandi D, et al. Modulation of telomerase expression and function by miRNAs: Anti-cancer potential. Life Sci 2020;259:118387 View Article PubMed/NCBI
  69. Zhou Z, Li Y, Xu H, Xie X, He Z, Lin S, et al. An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase. iScience 2022;25(2):103813 View Article PubMed/NCBI
  70. Chen S, Wang R, Peng S, Xie S, Lei C, Huang Y, et al. PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing. Chem Sci 2022;13(7):2011–2020 View Article PubMed/NCBI
  71. Liang J, Teng P, Hu L, He G, Song Q, Zhang Y, et al. Platinum nanoparticles (PtNPs)-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samples. Anal Chim Acta 2022;1225:340203 View Article PubMed/NCBI
  72. Jia Z, Li Z, Liu C. CRISPR-powered Biosensing Platform for Quantitative Detection of Alpha-fetoprotein by a Personal Glucose Meter. Sens Actuators B Chem 2023;390:133994 View Article PubMed/NCBI
  73. Yang M, Shi K, Liu F, Kang W, Lei C, Nie Z. Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection. Sci Chin Chem 2020;64(2):330–336 View Article PubMed/NCBI
  74. Zhao J, Tan Z, Wang L, Lei C, Nie Z. A ligation-driven CRISPR-Cas biosensing platform for non-nucleic acid target detections. Chem Commun (Camb) 2021;57(57):7051–7054 View Article PubMed/NCBI
  75. Chen Q, Tian T, Xiong E, Wang P, Zhou X. CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection. Anal Chem 2020;92(1):573–577 View Article PubMed/NCBI
  76. Liu L, Wang Z, Wang Y, Luan J, Morrissey JJ, Naik RR, et al. Plasmonically Enhanced CRISPR/Cas13a-Based Bioassay for Amplification-Free Detection of Cancer-Associated RNA. Adv Healthc Mater 2021;10(20):e2100956 View Article PubMed/NCBI
  77. Zhao X, Li S, Liu G, Wang Z, Yang Z, Zhang Q, et al. A versatile biosensing platform coupling CRISPR–Cas12a and aptamers for detection of diverse analytes. Sci Bull 2021;66(1):69–77 View Article PubMed/NCBI
  78. Chen H, Zhuang Z, Chen Y, Qiu C, Qin Y, Tan C, et al. A universal platform for one-pot detection of circulating non-coding RNA combining CRISPR-Cas12a and branched rolling circle amplification. Anal Chim Acta 2023;1246:340896 View Article PubMed/NCBI
  79. Wu YX, Xing S, Wang Y, Tian BY, Wu M, Wang XP, et al. Multiple TMA-aided CRISPR/Cas13a platform for highly sensitive detection of IL-15 to predict immunotherapeutic response in nasopharyngeal carcinoma. J Immunother Cancer 2023;11(8):e006552 View Article PubMed/NCBI
  80. Wang Q, Liu Y, Yan J, Liu Y, Gao C, Ge S, et al. 3D DNA Walker-Assisted CRISPR/Cas12a Trans-Cleavage for Ultrasensitive Electrochemiluminescence Detection of miRNA-141. Anal Chem 2021;93(39):13373–13381 View Article PubMed/NCBI
  81. Cui Y, Fan S, Yuan Z, Song M, Hu J, Qian D, et al. Ultrasensitive electrochemical assay for microRNA-21 based on CRISPR/Cas13a-assisted catalytic hairpin assembly. Talanta 2021;224:121878 View Article PubMed/NCBI
  82. Sun H-H, He F, Wang T, Yin B-C, Ye B-C. A Cas12a-mediated cascade amplification method for microRNA detection. Analyst 2020;145(16):5547–5552 View Article PubMed/NCBI
  83. Peng S, Tan Z, Chen S, Lei C, Nie Z. Integrating CRISPR-Cas12a with a DNA circuit as a generic sensing platform for amplified detection of microRNA. Chem Sci 2020;11(28):7362–7368 View Article PubMed/NCBI
  84. Bruch R, Baaske J, Chatelle C, Meirich M, Madlener S, Weber W, et al. CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics. Adv Mater 2019;31(51):e1905311 View Article PubMed/NCBI
  85. Albini A, Sporn MB. The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer 2007;7(2):139–147 View Article PubMed/NCBI
  86. Junttila MR, de Sauvage FJ. Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 2013;501(7467):346–354 View Article PubMed/NCBI
  87. Dhainaut M, Rose SA, Akturk G, Wroblewska A, Nielsen SR, Park ES, et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 2022;185(7):1223–1239.e20 View Article PubMed/NCBI
  88. Dervovic D, Malik AA, Chen ELY, Narimatsu M, Adler N, Afiuni-Zadeh S, et al. In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer. Nat Commun 2023;14(1):3150 View Article PubMed/NCBI
  89. Giuffrida L, Sek K, Henderson MA, Lai J, Chen AXY, Meyran D, et al. CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun 2021;12(1):3236 View Article PubMed/NCBI
  90. Lin M, Yang ZZ, Yang YL, Peng YW, Li JJ, Du YT, et al. CRISPR-based in situ engineering tumor cells to reprogram macrophages for effective cancer immunotherapy. NANO TODAY 2022;42:101359 View Article PubMed/NCBI
  91. Lu S, Yang N, He J, Gong W, Lai Z, Xie L, et al. Generation of Cancer-Specific Cytotoxic PD-1(-) T Cells Using Liposome-Encapsulated CRISPR/Cas System with Dendritic/Tumor Fusion Cells. J Biomed Nanotechnol 2019;15(3):593–601 View Article PubMed/NCBI

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Yang QL, Zhong XY, Zhong LM, Zhang H, Hu SW. Advances in CRISPR Based Biosensing Strategies for Cancer Diagnosis. Cancer Screen Prev. 2024;3(1):61-71. doi: 10.14218/CSP.2023.00026.
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Received Revised Accepted Published
October 31, 2023 November 22, 2023 December 20, 2023 March 25, 2024
DOI http://dx.doi.org/10.14218/CSP.2023.00026
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