Changes in genomic miRNA copy numbers and gene positions (amplification, deletion or translocation) are frequently related to the cause of abnormal miRNA expression in malignant cells (96). The loss of the miR-15a/16-1 cluster gene at chromosome 13q14, which is commonly seen in individuals with B-cell chronic lymphocytic leukemia, is the earliest example of a change in miRNA gene placement (97). In addition, the 5q33 region that contains miR-143 and miR-145 is frequently deleted in lung cancer, which lowers the expression levels of both miRNAs (96). On the other hand, B-cell lymphoma (98) and lung cancer (99) exhibit amplification of the miR-17-92 cluster gene, and T-cell acute lymphoblastic leukemia (100) exhibits translocation of this cluster gene, resulting in upregulation of these miRNAs in these cancer types.

High-resolution array-based comparative genomic hybridization has been used to confirm the high frequency of genomic changes in miRNA loci in 227 human cancer samples of ovarian cancer, breast cancer and melanoma (101). Numerous miRNA genes are found in genomic areas linked to cancer, according to additional genome-wide studies (102,103). These areas on chromosomes may be fragile sites, common breakpoint regions or minimal regions of amplification, which may include oncogenes, or minimal regions of loss of heterozygosity, which may carry tumor suppressor genes (102). Considering all the observations, the findings suggest that the amplification or deletion of specific genomic regions containing miRNA genes may be the cause of abnormal miRNA expression in malignant cells.

Since a number of transcription factors tightly regulate miRNA production, dysregulation of some important transcription factors, including p53 and c-Myc, may be linked to aberrant miRNA expression in cancer (104,105). By binding to E-box regions in the miR-17-92 promoter, c-Myc is commonly increased through transcriptional activation, in a number of malignancies to control cell proliferation and death, and promotes the transcription of the oncogenic miR-17-92 cluster (106). In keeping with its carcinogenic function, c-Myc also inhibits the transcriptional activity of tumor-suppressive miRNAs, including the let-7, miR-26, miR-29 and mir-15a families (107).

In hepatocellular carcinoma (HCC), c-Myc and the tumor suppressor miR-122 are reciprocally regulated. By attaching itself to its promoter, c-Myc inhibits the synthesis of miR-122 and, by targeting the transcription factor Dp2 and the transcription factor E2F transcription factor 1 (E2f1), indirectly stops c-Myc transcription. Therefore, the disruption of this feedback loop between miR-122 and c-Myc is necessary for the development of HCC (108). HCC is brought on by direct binding of c-Myc to the promoters of the miR-148a-5p and miR-363-3p genes, which inhibits their synthesis and encourages the G₁ to S phase transition. miR-148a-5p, on the other hand, directly targets and suppresses the production of c-Myc, while miR-363-3p destabilizes c-Myc by targeting ubiquitin-specific protease (106,109). One of the most frequently altered genes in human cancer is TP53, which encodes the tumor suppressor p53. Another manner in which transcriptional factors control miRNA expression to exert a tumor suppressive function is the p53-miR-34 regulatory axis (110). A complex p53 network that controls cell-cycle progression and apoptosis is formed by the p53-regulated expression of several genes, including miRNA genes. Similar to p53-mediated phenotypes, the miR-34 family, comprising miR-34a, miR-34b and miR-34c, causes cell-cycle arrest, cell senescence and apoptosis in cancer (111), suggesting that p53 and miR-34 are involved in the same regulatory mechanism.

Additional research has revealed that p53 regulates the expression of several miRNAs, including miR-605 (112), miR-1246 (113) and miR-107 (114), to carry out its role. Additional transcriptional factors have been identified to control miRNA production in addition to the two most well researched transcriptional factors, c-Myc and p53. For instance, miR-223 is inhibited in a variety of cancer types, including HCC and acute myeloid leukemia (AML), and it is predominantly expressed in the hematopoietic system, which serves important roles in the formation of myeloid lineages (115).

As aforementioned, several enzymes and regulatory proteins, including Drosha, Dicer, DGCR8, AGO and XPO-5, intricately regulate miRNA biogenesis, enabling proper maturation of miRNA from pri-miRNA precursors. Therefore, aberrant expression of miRNAs may result from mutations or aberrant expression of any protein in the miRNA biogenesis pathway. Two important RNase III endonucleases in miRNA maturation, Drosha and Dicer, oversee the creation of pre-miRNA and the miRNA duplex (116,117). According to previous studies, both enzymes are dysregulated in some cancer types such as breast and bladder cancer (118,119).

A portion of miRNAs are controlled during the Drosha-processing stage, and this control affects miRNA expression in cancer and during embryonic development (120). It has been reported that 15% of 534 Wilms tumors had single-nucleotide substitution/deletion mutations in DGCR8 and Drosha, which resulted in reduced expression of mature Let-7a and the miR-200 family (121). In terms of Dicer dysregulation, it has been noted that CRC cells with impaired Dicer acquire a higher propensity for tumor initiation and metastasis (122). Furthermore, patients with ovarian cancer with higher Dicer and Drosha mRNA levels have a higher median survival rate (123). On the other hand, patients with lower Dicer expression have a considerably lower survival rate (124,125).

Reduced let-7 expression and poor postoperative survival have been linked to lower Dicer mRNA levels in patients with lung cancer (126). As with Dicer and Drosha, cancer also leads to deregulation of AGO. For instance, kidney tumors caused by Wilms disease frequently exhibit loss of the human eukaryotic initiation factor 2C1/human argonaute gene (127). Human AGO proteins are crucial in regulating cell-dependent gene expression. AGO2 expression levels, for example, are considerably higher in basic gastric cancer and the lymph node metastases (128), but AGO2 expression is lower in melanoma, which corresponds to a lower RNA interference efficiency, compared with in primary melanocytes (129).

A fraction of human cancers with microsatellite instability have inactivating mutations in the XPO-5 gene. The insertion of an 'A' in exon 32 in CRC cells (HCT-15 and DLD-1 cells), results in an early termination codon, which causes a frameshift mutation and the creation of a shortened protein. This shortened XPO-5 is no longer able to export pre-miRNAs. As a result, there is less miRNA processing since pre-miRNAs are confined to the nucleus. The restoration of XPO-5 activities possesses tumor-suppressor properties and restores the defective export of pre-miRNAs (130). It has also been noted that ERK phosphorylates XPO-5, preventing XPO-5 from moving pre-miRNAs from the nucleus to the cytoplasm in HCC (131).

A well-known characteristic of cancer is epigenetic modification, which includes the disruption of histone modification, aberrant DNA hypermethylation of tumor suppressor genes and widespread genomic DNA hypomethylation. Similar to protein coding genes, miRNAs are considered to be subjected to epigenetic modifications (132,133). For example, it has been reported that acute myeloid leukemia 1/eight twenty one, the most prevalent AML-associated fusion protein, epigenetically suppresses miR-223 expression via CpG methylation (134). A study reported that the expression levels of 17 out of 313 human miRNAs were more than three times higher in T24 bladder cancer cells after concomitant treatment with DNA methylation and histone acetylation inhibitors (134).

The downregulation of the proto-oncogene BCL6 coincides with markedly increased expression of miR-127, which is embedded in a CpG island and not expressed in cancer cells (135). These findings suggest that miRNAs may function as tumor suppressors and can be activated through DNA demethylation and histone deacetylase inhibition. In addition, it has been reported that miR-148a and the miR-34b/c cluster are susceptible to hypermethylation-associated silencing in cancer cells (136). Furthermore, in vivo metastasis formation was suppressed, tumor growth was decreased and cancer cell motility was inhibited when these miRNAs were restored (137). Similarly, DNA hypermethylation has been linked to lower expression levels of miR-9-1, miR-124a and miR-145-5p in colon, lung and breast cancer, respectively (138,139). This suggests that the abnormal histone acetylation and DNA methylation of miRNA genes could be useful indicators for the diagnosis and prognosis of cancer. These findings emphasize the significance of epigenetic control in miRNA production during carcinogenesis.

The primary cause of tumorigenesis is aberrant cell proliferation, which is the most significant characteristic of cancer. Certain miRNAs functionally incorporate into several cell proliferation pathways, and the dysregulation of these miRNAs directs the cancer cells to continue proliferative signaling to avoid growth suppressors. miRNAs serve a role in controlling the expression of E2F proteins (cell proliferation regulators). E2F1-deficient animals develop a wide range of malignancies, and this member of the E2F family is characterized as a tumor suppressor and stimulates target gene transcription during the G₁ to S transition (141). miR-17-92, once activated by c-Myc, suppresses the translation of E2F1 (106). E2F1 protein levels may not increase markedly in response to c-Myc activation if the miR-17-92 cluster acts as a brake on this putative positive feedback loop, given that c-Myc also directly stimulates E2F1 production (142). Furthermore, the miR-17-92 cluster has been demonstrated to regulate E2F2 and E2F3 translation (143). Additionally, the expression of the miR-17-92 cluster might be triggered by the E2F transcription factors (144). Therefore, under normal conditions, the feedback loop between E2F and the miR-17-92 cluster provides a means of preserving regular cell-cycle progression. However, miR-17-92 upregulation, which is common in several cancer types such as colorectal cancer and gastric cancer, disrupts the feedback loop that encourages cell proliferation (145).

Different cyclins, Cdks and their inhibitors, extensively regulated by miRNAs, are essential for cell-cycle progression. The G₁/S transition inhibition of Drosophila germline stem cells with Dicer-1 deletion suggests that miRNAs are required for germline stem cells to pass through the normal G₁/S checkpoint (146). Additionally, Dacapo, a member of the p21/p27 family of Cdk inhibitors, is upregulated in Dicer-deficient germline stem cells, suggesting that miRNAs negatively control this protein to encourage cell-cycle progression (147). In glioblastoma cells, miR-221/222 has been found to directly target the Cdk inhibitor p27Kip1 (148), a finding that has been subsequently validated in original tumor samples and other cancer cell lines (149,150). While its inhibition causes G₁ cell-cycle arrest in malignant cells, ectopic expression of miR-221/222 enhances cell proliferation. Furthermore, several human malignancies have been reported to be associated with increased expression levels of miR-221/222, suggesting that regulation of p27Kip1 by miR-221/222 is a valid carcinogenic pathway (151).

To meet the demands of food and oxygen during tumor growth and metastasis, the highly coordinated process of angiogenesis produces new blood vessels from pre-existing ones (152). Because the oxygen concentration of tumor tissues is lower than that of the surrounding normal tissues, hypoxia serves a critical role in the tumor microenvironment by facilitating the proliferation and maintenance of cancer cells. In response to hypoxia, a crucial transcription factor, hypoxia-inducible factor (HIF), affects the expression of several genes, including miRNAs (153). As a key angiogenic factor, VEGF instructs endothelial cells to form new vessels when it binds to its receptors (154). Therefore, angiogenesis is likely to be impacted by miRNAs that target the VEGF or HIF signaling pathways.

It is widely known that miRNAs intricately govern the angiogenesis process (155,156). The type of miRNA most frequently increased during hypoxia is miR-210 (157). In normoxic human umbilical vein endothelial cells, miR-210 overexpression stimulates the formation of capillary-like structures and VEGF-dependent cell migration. miR-210 blockage, on the other hand, inhibits these mechanisms (158,159). Furthermore, miR-210 stimulates angiogenesis by upregulating the expression levels of VEGF and its receptor, in addition to targeting the antiangiogenic factor receptor tyrosine kinase ligand ephrin-A3 (159).

Hypoxia causes endothelial cells to produce miR-424, which targets cullin 2, a ubiquitin ligase scaffold protein, to stimulate angiogenesis. By stabilizing HIF-1α, this step enables stabilized HIF-1α to transcriptionally trigger the expression of VEGF (160). miR-21 is another miRNA that promotes angiogenesis. To activate the downstream Akt/ERK signaling pathways and increase HIF-1α and VEGF expression, it targets PTEN (161). By inhibiting VEGF and/or HIF-1α, on the other hand, miR-20b and miR-519c negatively control angiogenesis (162,163). The downregulation of miR-107 increases tumor angiogenesis under hypoxic conditions because it not only regulates HIF-1α but also inhibits the production of HIF-1β (114).

The biology of metastasis is an intricate, multi-step and dynamic process. The loss of cell adhesion due to E-cadherin suppression and the activation of genes linked to invasion and motility are the hallmarks of the EMT, which is regarded as an initial and crucial stage of metastasis (164). Numerous miRNAs alter the expression of genes, including MMPs and their inhibitors, that control extracellular matrix (ECM) turnover and breakdown (165). Proliferation, migration, differentiation and apoptosis are among the cell phenotypic alterations that are influenced by ECM turnover or remodeling (166). Zinc-dependent endopeptidases, known as important MMPs such as MMP-1, -2, -3, -7 and -9, are crucial for cell migration, adhesion, dispersion, differentiation and ECM modeling (167). They can cause cell surface receptors to cleave ECM proteins such as type IV collagen, interestitial collagen, elastin and casein for degradation (168). Exogenous miR-143 expression in osteosarcoma cells leads to downregulated MMP-13 protein levels and reduced cell invasion (169). In clinical samples, MMP-13 was found in cases with lung metastases and low miR-143 expression, while it was not found in patients without metastases and with high miR-143 expression (170).

It has been revealed that miR-206 decreased the amount of the cell division cycle 42, MMP-2 and MMP-9 proteins in human breast cancer (171). Due to the control of actin cytoskeleton remodeling, including filopodia formation, this protein level regulation inhibited MDA-MB-231 cell invasion and migration (171). Furthermore, downregulation of miR-340 has been associated with aggressive activity in several breast cancer cell lines (172). Because it directly targets c-Met, the stimulation of miR-340 expression suppresses the motility and invasion of breast tumor cells, thereby modulating the production of MMP-2 and MMP-9 (172).

Numerous signaling pathways, including the TGF-β pathway, are considered to govern EMT. These pathways converge on important transcription factors, including Zinc finger E-box-binding homeobox (ZEB), SNAIL and TWIST (173). TGF-β-regulated miRNAs participate in TGF-β signaling to trigger EMT and promote metastasis in advanced cancer. miR-155 is implicated in this regulation, is transcriptionally activated by TGF-β/SMAD4 signaling and is upregulated in several malignancies such as colorectal and breast cancer (174). Furthermore, miR-155 stimulates EMT by targeting RhoA GTPase, an essential modulator of cellular polarity and the formation and maintenance of tight junctions. Additionally, miR-155 suppression prevents TGF-β-induced EMT, tight junction disruption, cell migration and invasion (174). In contrast to miR-155, TGF-β inhibits miR-200 and miR-203, and it has been demonstrated that the miR-200 family influences EMT by suppressing the expression of the transcriptional repressors (ZEB1 and ZEB2) of E-cadherin (175). ZEB1 and ZEB2, in turn, also suppress the miR-200 transcript, creating a double-negative feedback loop between the miR-200 family and ZEB1/ZEB2 (176).

An important characteristic of tumor growth is evasion of apoptosis, which is considered to be controlled by miRNAs (177,178). Different strategies are developed by tumor cells to prevent or delay apoptosis. The most prevalent of these is the loss of p53 tumor suppressor activity (179). Other strategies to avoid apoptosis include the suppression of proapoptotic proteins, upregulation of anti-apoptotic regulators and blocking the death pathways (180). miRNAs either inhibit or activate the components involved in anti-apoptosis mechanisms. It has been demonstrated that numerous p53-regulated miRNAs contribute to p53 functions; some of these miRNAs can feedback-modify p53 activity (181). For example, in multiple myeloma, p53 transcriptionally activates three miRNAs (miR-192, miR-194 and miR-215) to bind directly with the mRNA of mouse double minute 2 homolog (Mdm2) and prevent p53 from being destroyed, thus inhibiting Mdm2 expression (182). The development of multiple myeloma is influenced by the downregulation of these miRNAs, which are positive regulators of p53 (183).

Another negative feedback regulation takes place between p53 and miR-122 by targeting cyclin G1 (184) and cytoplasmic polyadenylation element-binding protein (185). The creation of a chemotherapy and miRNA-based therapeutic combination for HCC is made possible by the stimulation of p53 activity by miR-122, which also increases cell sensitivity to doxorubicin (186). In addition, cancer cells are resistant to death due to other dysregulated p53-regulated miRNAs. For example, the miR-17-92 cluster represents a novel target for p53-mediated transcriptional repression during hypoxia. While its upregulation prevents apoptosis, its downregulation makes cells more vulnerable to hypoxia-induced apoptosis. Consequently, tumor cells that express more miR-17-92 may be able to evade the apoptosis caused by hypoxia (187). All these aforementioned findings demonstrate that, in healthy circumstances, p53 and miRNAs regulate a network that intricately determines cell destiny. However, dysregulation of p53 or its target miRNAs may allow cancer cells to evade cell death.

Some miRNAs that serve a role in cell death may target proapoptotic factors (Bax, Bim and Puma) and anti-apoptotic regulators (Bcl-2 and Bcl-xL). In chronic lymphocytic leukemia, miR-15a and miR-16-1 are downregulated, and their expression is inversely associated with that of Bcl-2 (97). Additional research revealed that these two miRNAs caused apoptosis and suppressed Bcl-2 expression (188). Other miRNAs, including miR-204 (189), miR-148a (113) and miR-365 (190), also control Bcl-2 expression. miR-491-5p effectively causes ovarian cancer cells to undergo apoptosis by causing Bim accumulation and directly suppressing Bcl-xL expression (191).

The role of some miRNAs and their relationship with tumor occurrence, development, metastasis and survival is shown in Table I.

The Cas13 protein of Leptotrichia buccalis consists of a single effector molecule (~1,159 aa residues) and a 64-66 nucleotide crRNA, which make up the CRISPR/Cas13 system (198,199) (Fig. 4). The Cas13a type protein crystal structure along with the crRNA secondary structure has been studied in more detail compared with that of other Cas13 (b-d, x and y) types (200,201). The Cas13a protein, isolated from the Gram-negative bacterium Leptotrichia shahii, has been identified to catalyze two reactions: One that matures crRNA and the other that cleaves the target RNA (202). In this single effector protein, two domains, the nuclease (NUC) domain and the crRNA recognition (REC) lobe, make up the entire molecule (Fig. 4A). The N-terminal domain (NTD) and a domain known as helix-1 are both present in the REC lobe (202). The non-conserved area of Cas13a known as the NTD is made up of two subdomains: A smaller subdomain with three α-helices, a β-hairpin and a β-sheet, and a bigger subdomain with an ordered fragment made up of seven α-helices and a disordered fragment (203) (Fig. 4).

The V-shaped structure of the helix-1 domain is formed by seven α-helices. The crRNA binding channel is formed by the positively charged surface of the helix-1 domain facing the NTD domain (204). The two conserved higher eukaryotic and prokaryotic nucleotide-binding (HEPN) domains (HEPN-1 and HEPN-2), a linker that joins the two HEPN domains, and a helix-2 domain are all found in the NUC lobe (202). The helix-1 and HEPN domains are responsible for the two enzymatic functions of Cas13a. The helix-2 domain further sub-divides the HEPN-1 domain into two subdomains. Three α-helices make up the HEPN 1-II subdomain, whereas four α-helices and a brief β-hairpin make up the HEPN-1 I subdomain (Fig. 4B). Seven α-helices plus a double-stranded β-sheet directly make up the HEPN2 domain structure (202). Between the two HEPN-1 subdomains lies the helix-2 domain, which is made up of eight bean-shaped α-helices (204). The Cas13a/crRNA complex targets the matching RNA under the direction of crRNA, triggering the protection of prokaryotes against RNA viruses (205,206) (Fig. 4F and G).

The structure of the crRNA is composed of a spacer sequence [guide RNA (gRNA)] that mediates target recognition by RNA-RNA hybridization and a repeat stem-loop region (referred to as the 5′-handle) that prevents this region from being cleaved during the cleavage of the target RNA by Cas13a (207) (Fig. 4C). The stem-loop is composed of two single-stranded bases at the base, a nine-base loop, neighboring motifs at both ends and a stem made up of five base pairings. The repeat region of crRNA can twist slightly to generate a helical shape in addition to a stem-loop secondary structure (202). This structure is primarily sustained by the creation of strong hydrogen bonds between the HEPN2 domain and the backbone of the crRNA (204). It is possible to cause the Cas13a protein to recognize the stem-loop structure of the crRNA in a sequence-specific way by altering stem nucleotides, which is necessary for its nuclease cleavage activity (204) (Fig. 4D).

Within the crRNA guide region, one to four nucleotides oversee the 5′-end being attached to the void created by the HEPN-1 and helix-2 domains, making a U-turn. The linker and the groove created by the HEPN-2 domain bind the final three or four nucleotides (16). To identify the target RNA, the Cas13 protein buries a spacer of eight or nine nucleotides at the 5′-end, while the middle region and 3′-end are exposed to the peripheral solvent (204). To determine the ideal gRNA, researchers have created a computational model, and identified that the properties of crRNA and the environment of the target RNA are important constraints on the cleavage effectiveness of Cas protein by assessing the activities of 24,460 gRNAs and looking for mismatches between gRNAs and the target sequence (208).

Cas13 action does not require crRNA maturation; in fact, pre-crRNA can detect target RNAs (also referred to as activator RNA; Fig. 4E) (209). The ribonucleoprotein complex (RNP) undergoes a conformational change when crRNA binds to a target RNA (Fig. 4F). A catalytic site is formed in part by the close interaction of two HEPN domains (201). The binding of crRNA is anticipated to result in the cleavage of the target RNA as well as other ssRNAs surrounding the RNP complex, possibly including host RNA, because of the distance between the catalytic site and the crRNA-RNA duplex. This activity of the CRISPR/Cas13 system, known as collateral damage or collateral cleavage, refers to the off-target cleavage of endogenous RNA (205,210).

According to one study, sequence-specific activation of non-specific RNA cleavage may help to protect the nearby cells by causing cell dormancy or death, or it may improve the prevention of phage replication by removing all RNA from a cell in bulk (195). This ability has been widely used in RNA knockout strategies, disease treatment, interference with viral infection, screening of loss of function mutants, molecular detection of biological agents and CRISPR-based antimicrobials (211-213). Therefore, the CRISPR/Cas13 system can simultaneously cleave target RNA and non-target RNA (206,214). Furthermore, in Cas13, the mutation of arginine in the HEPN domain, which is responsible for RNA cleavage, results in the formation of catalytically inactive Cas13, known as dead Cas13 (dCas13). This form of Cas13 has been used to increase the application of Cas13 in RNA research. Through the binding of fluorescent proteins or enzymes, mutant dCas13 persistently attaches to target RNA and offers a platform for detecting transcripts in living cells (20,215).

Colorimetric assays typically examine the color shift within the test solution. Without the need for complex equipment, miRNA detection can be performed quickly and easily with the use of a colorimeter or the human eye (226). Once Cas12a/crRNA, Cas13a/crRNA or Cas14/crRNA identify their target DNA or RNA, they exhibit a transition to the active state, where their collateral cleavage activity cleaves the ssRNA or ssDNA substrates (227,228). With the aid of CRISPR/Cas recognition for miRNA sensing, researchers have created a DNA probes and gold nanoparticles (AuNPs)-based platform. Cas12a/crRNA's programmable recognition of DNA and Cas13a/crRNA's programmable recognition of RNA initiates ssRNA or trans-ssDNA cleavage of their corresponding complementary target (229). For the intended AuNPs-DNA probe pair, target-induced trans-ssDNA or -ssRNA cleavage results in a change in aggregation behavior, which allows direct visual observation in an hour of various samples, including miRNAs (229). For naked-eye gene detection platforms, a universal linker ssRNA or ssDNA acts as a trans-cleavage substrate for the CRISPR/Cas13a or CRISPR/Cas12a systems. Furthermore, two universal AuNPs DNA probes have been created to hybridize with either ssRNA or linker ssDNA (229).

Since trans-cleavage is not triggered in the absence of a target (such as miRNA sequences), the linker ssDNA or ssRNA stays intact during the reaction. An aggregated state is created by hybridization-induced cross-linking of the AuNPs-DNA probe pair (229). After the trigger of trans-cleavage activity, the linker nucleotides are broken down once the CRISPR/Cas systems identify their target nucleotides. By identifying cross-linked and scattered AuNPs-DNA probes (229), visual detection can be approximated (Fig. 5B). The sensitivity of miRNA detection is as low as 500 fM, which is on par with or better than that of techniques based on dual-labeled fluorescent ssRNA reporters (229). When comparing 1.0 nM target miRNA-17 with miRNA-10b, miRNA-21 and miRNA-155 by colorimetry at the same concentration, this approach demonstrated a strong level of specificity (229). Furthermore, family miRNA members with highly related sequences, such as miRNA-17, miRNA-20a, miRNA-20b and miRNA-106a, can be distinguished by single- or double-nucleotide variations at a concentration of 1.0 nM using CRISPR/Cas-based colorimetric assays (229).

The Leptotrichia buccalis Cas13a (LbuCas13a) protein has been utilized to directly detect miRNAs with great specificity and simplicity compared with stem-loop RT-PCR, which requires three oligonucleotides, a cDNA synthesis step and different temperature cycles to replicate amplicons (232). To achieve an enhanced fluorescence signal, a crRNA and moderate isothermal conditions are used. According to the fluorescence spectroscopy results, the poly-U RNA probe (fluorescent quencher 5′ U) labeled with fluorophore (6-FAM) and quencher (BHQ1) is successfully cleaved when introduced into the miRNA detection test, resulting in fluorescent signals (233) (Fig. 5D). Based on the trans-ribonuclease activity of Cas13a and base pairing between crRNA and miR-17 (selected as the model target), researchers have able to detect the miR-17 at as low as 4.5 aM concentration within 30 min (233). Furthermore, rationally programmed crRNA is able to direct the LbuCas13a/crRNA complex to distinguish single-nucleotide changes, even at the end of target miRNA (234). The relative measurement of miR-17 in four different cell line samples, including three human breast cancer cell lines, validated the uniqueness and validity of the approach (233). The high sensitivity of identifying miRNAs in total small RNA preparations from serum samples also suggests a useful application of the one-step Cas13a/crRNA-based detection approach for miRNA-related disease diagnostics (233).

A novel approach known as dd-Cas13a increases the concentration of local molecules for an effective reaction or detection (234). Researchers have adopted a droplet microfluidic technology to increase the local concentration of the RNA reporter and target sequence for the Cas13a system in cell-sized volumes (235,236). Thousands of picolitre-sized droplet reactors are filled with an oil-emulsified combination of target RNAs and Cas13a. Such droplets can be illuminated by the cumulative fluorescence signal from the collateral cleavage of a single RNA target-activated Cas13a (235,236) (Fig. 5E). Under these circumstances, a single-target RNA can cause the cleavage of >100 quenched fluorescent RNA reporters following recognition by a crRNA, producing a fluorescence-positive droplet. Therefore, dd-Cas13a eliminates the necessity for reverse transcription and amplification, and enables absolute digital quantification of single unlabeled RNA molecules (235,236). The utility of the dd-Cas13 assay was assessed by measuring miRNA-17 expression in several cell lines, such as human glioma cells, normal human breast cells and adenocarcinoma cells, and 100% results that corresponded with RT-qPCR data were obtained (235).

Based on the ECL approach and employing the trans-cleavage activity of CRISPR/Cas13a, scientists have engineered a sophisticated platform known as PECL-CRISPR, which mediates the exponential amplification of the ultrasensitive detection of miRNAs that differ even by a single-base (232).

Quantitative miRNA detection is made possible by the ECL signal on a bipolar electrode (BPE), which directly reflects the oxidation reaction of the ruthenium (II) polypyridyl complex [Ru(phen)2dppz]2⁺ and DNA at the BPE anode, and is directly related to the miRNA concentration (232) (Fig. 5G). By identifying miR-17 from various human cancer cells, the application of PECL-CRISPR has been examined, proving that the platform has great potential for miRNA diagnosis (232).

By logically designing the crRNA, the platform exhibits the ability to differentiate between highly homologous members of the miR-17 and let-7 families by introducing a mismatch at the precise location of the crRNA, independent of the single distinct base locations at the 5′ or 3′ end of the target (233). The LOD of this platform for miR-17 was 1.0×10⁻¹⁵ M, which is approximately three orders of magnitude lower than that of the direct detection of miR-17 (LOD, 1.0×10⁻¹² M) using LbuCas13a (233).

For on-site detection of miRNAs, a CRISPR/Cas13-based biosensor has been created as a user-friendly and cheaper electrochemical microfluidic platform (EM-CRISPR/Cas13a), which substitutes a Cas13a-driven signal amplification for synthetic nucleic acid amplification steps (238). In this assay, a streptavidin is applied to the chip inlet to functionalize the immobilized surface area of the biosensor for the CRISPR/Cas13a-powered miRNA detection. All unbound biomolecules are eliminated by vacuuming the channel inlet (238). To activate Cas13a and perform the collateral cleavage of the reporter RNA (reRNA), the samples (biotin and 6-FAM-tagged reRNA), and the other sample (containing the target miRNA and the Cas13a effector with its target-specific crRNA) are mixed separately (238) (Fig. 5H).

An enzymatic readout of the assay is made possible by the addition of anti-fluorescein antibodies bonded with glucose oxidase (GOx), which bind only to the uncleaved reRNAs (Fig. 5H). Since GOx catalyzes its substrate to produce H₂O₂, which is amperometrically measured in the electrochemical cell, a glucose solution is used in this assay for the readout (238). Without performing nucleic acid amplification, researchers have used this new combination to detect the miRNA levels of the putative brain tumor markers miR-19b and miR-20a in serum samples. With a setup time of <4 h and a readout time of 9 min, the EM-CRISPR/Cas13a biosensor achieved a detection limit of 10 pM with a measuring volume of <0.6 liters. Additionally, this biosensor platform was able to identify miR-19b in serum samples of children with brain cancer, proving that this electrochemical CRISPR-powered system is a low-cost, readily scalable and target amplification-free molecular approach for miRNA-based diagnostics (238).

The development of diagnostic tools and techniques for multiplexing approaches is based on analyzing the abundance of a number of specific components in individual samples from a patient. However, the variety of Cas protein types and the potential for cross-reactions between distinct Cas13a proteins may restrict the use of Cas13a (239).

This might be resolved using a multichannel microfluidic chip technique.

Researchers have developed various multiform versions of electrochemical microfluidic biosensors by segmenting the channels into subsections, using the same principle as the EM-CRISPR/Cas13a system (Fig. 5H). This resulted in four unique chip designs for the simultaneous quantification of a maximum of eight miRNAs using the innovative system called CRISPR-Biosensor X. Without altering the sensor or measurement configuration, this system can work on a single clinical sample with a single effector protein (239).

Because of their high sensitivity, selectivity and low instrument costs, electrochemical biosensors are emerging as a potent detection technique platform (240). Using the CRISPR/Cas13a platform in conjunction with the catalytic hairpin assembly (CHA) process, an ultrasensitive electrochemical biosensing platform has been designed for miRNA-21 detection (241). CHA is a high-efficiency, isothermal, enzyme-free amplification technique that may be used in a variety of analytical formats, such as electrophoretic (242), colorimetric (243), fluorescence (231), surface plasmon resonance (244) and electrochemical approaches (245).

This electrochemical biosensor exhibited a strong biosensing activity from 10 fM to 1.0 nM with an LOD of 2.6 fM using the CRISPR/Cas13a-mediated cascade signal amplification approach (241). The use of the biosensing platform for miRNA-21 detection was assessed. A clinical sample (human serum; diluted ten times), was used to test the analytical reliability before being tested for various concentrations of miRNA-21 (10 fM to 1.0 nM). The electrochemical recoveries were ~98.2%, demonstrating that the CRISPR/CHDC assay can be used to identify miRNA-21 in biological materials (241).

The off-target consequences of the CRISPR/Cas13a system are minimal. One to two mismatches in the core of a gRNA diminish or eliminate its activity, enabling a mismatch control for each targeting gRNA (20,246). In this context, the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection system, employs the CRISPR/Cas13a system as a quick DNA or RNA detection technique with aM (10⁻¹⁸ M) sensitivity and single-base mismatch specificity (247). This approach uses isothermal RPA, transcription and detection by Cas13 platforms (247). The signal can be recorded on a colorimetric lateral-flow strip or can be observed by fluorescence signal readout to facilitate the rapid identification of most types of suitable Cas13 RNA target. SHERLOCK enables single base-pair mismatch specificity, quick (setup time, <15 min) and accurate nucleic acid detection at concentrations as low as ~2 aM (248).

A summary of CRISPR/Cas13 systems used in miRNA detection platforms is shown in Table III.