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Cas13a-based nucleic acid amplification-free biosensors. (A) Schematic of Cas13a-based biosensor using combination of crRNAs. Two or three crRNAs targeting distinct regions of SARS-CoV-2 genome are mixed to increase the sensitivity of Cas13a-based detection. (B) Schematic of ultralocalized Cas13a assay. The Cas13a reaction is confined in cell-like-sized reactor to enhance the local reaction concentration. (C) Schematic of opn-SATORI. In this detection, biotin-labeled Cas13a complexes are captured by streptavidin-labeled magnetic beads, which are concentrated by the magnetic force into microchambers and are activated in the presence of target RNAs. (D) Schematic of casCRISPR. The trans-cleavage activities of both Cas14a and Cas13a reactions are orderly combined to detect targets.
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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a syste...
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria conta...
CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-p...
Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manip...
A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking...
The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with C...
Citations
... 199 Moreover, RNA editing technologies such as CRISPR-based approaches can also be applied to modify and increase circRNA expression. 192,200,201 Regulating the expression of piRNAs is also a complex area of study. However, some methods have been proven to successfully regulate piRNA expression. ...
The utilization of PD-1/PD-L1 inhibitors marks a significant advancement in cancer therapy. However, the efficacy of monotherapy is still disappointing in a substantial subset of patients, necessitating the exploration of combinational strategies. Emerging from the promising results of the KEYNOTE-942 trial, RNA-based therapies, particularly circRNAs and piRNAs, have distinguished themselves as innovative sensitizers to immune checkpoint inhibitors (ICIs). These non-coding RNAs, notable for their stability and specificity, were once underrecognized but are now known for their crucial roles in regulating PD-L1 expression and bolstering anti-cancer immunity. Our manuscript offers a comprehensive analysis of selected circRNAs and piRNAs, elucidating their immunomodulatory effects and mechanisms, thus underscoring their potential as ICIs enhancers. In conjunction with the recent Nobel Prize-awarded advancements in mRNA vaccine technology, our review highlights the transformative implications of these findings for cancer treatment. We also discuss the prospects of circRNAs and piRNAs in future therapeutic applications and research. This study pioneers the synergistic application of circRNAs and piRNAs as novel sensitizers to augment PD-1/PD-L1 inhibition therapy, demonstrating their unique roles in regulating PD-L1 expression and modulating immune responses. Our findings offer a groundbreaking approach for enhancing the efficacy of cancer immunotherapy, opening new avenues for treatment strategies. This abstract aims to encapsulate the essence of our research and the burgeoning role of these non-coding RNAs in enhancing PD-1/PD-L1 inhibition therapy, encouraging further investigation into this promising field.
... The protein (Cas) 13a that is related to clustered regularly interspaced short palindromic repeat (CRISPR) has been produced as a very sensitive diagnostic method for in vitro nucleic acid identification. This is achieved via the non-specific RNA cutting capability of Cas13a [13]. The system relies on RNA fluorescence reporting probes, which are cut to emit fluorescence when Cas13a is activated by target RNA. ...
Mycobacterium bovis (M. bovis), the microorganism responsible for bovine tuberculosis (bTB), is transferred to people by the ingestion of unpasteurized milk and unprocessed fermented milk products obtained from animals with the infection. The identification of M. bovis in milk samples is of the utmost importance to successfully prevent zoonotic diseases and maintain food safety. This study presents a comprehensive description of a highly efficient molecular test utilizing recombinase-aided amplification (RPA)–clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) 13a–lateral flow detection (LFD) for M. bovis detection. In contrast to ELISA, RPA–CRISPR–Cas13a–LFD exhibited greater accuracy and sensitivity in the detection of M. bovis in milk, presenting a detection limit of 2 × 100 copies/μL within a 2 h time frame. The two tests exhibited a moderate level of agreement, as shown by a kappa value of 0.452 (95%CI: 0.287–0.617, p < 0.001). RPA–CRISPR–Cas13a–LFD holds significant potential as a robust platform for pathogen detection in complex samples, thereby enabling the more dependable regulation of food safety examination, epidemiology research, and medical diagnosis.
... Gene editing technology has been developing rapidly, among which CRISPR-Cas technology is considered to be the easiest and most powerful gene editing tool. Compared to the complex operation and high cost of ZFN and TALEN, CRISPR technology eliminates the tedious process of synthesizing and assembling protein modules that recognize DNA, and its guide RNA (gRNA) design and synthesis work is much less than the previous two and has low cytotoxicity (Zhao et al., 2022). Therefore, it has been widely applied immediately once introduced. ...
... The discovery that Cas13a effector is characterized by collateral RNA cleavage activity has led to greater interest in developing novel biosensing technologies for nucleic acid detection and holds the promise of significant advances in CRISPR diagnostics (Zhao et al., 2022). CRISPR-Cas13a systems are used to detect a variety of pathogens, such as bacteria, viruses, and cancer cell detection. ...
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.
... Cas13a randomly cuts adjacent RNA after its collateral-cutting capabilities is activated. This motivates Cas13a to create novel RNA-targeting tools and expand the application of CRISPR systems for gene detection (Abudayyeh et al., 2016;Zhao et al., 2022). By labeling RNA probes with fluorophores at both ends, CRISPR/Cas13a can detect RNA templates and amplify signals to achieve specific detection of target molecules (Abudayyeh et al., 2016;Kellner et al., 2019). ...
The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.
... CRISPR-Cas13a has been utilized in a variety of applications, including molecular diagnostics, gene therapy, gene editing, and RNA imaging [17][18][19][20][21], because it catalyzes cis-cleavage reactions on a specific single-stranded RNA (ssRNA) sequence containing a protospacer flanking site (PFS), followed by indiscriminate trans-cleavage reactions on ssRNA sequences [22,23]. T7 RNA polymerase (T7 RNAP) is a DNA-dependent RNA polymerase that produces RNA transcripts from template DNA with a T7 promoter and has been used in many studies including molecular diagnostics and RNA imaging [24][25][26][27]. ...
Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers.
... The HEPN domain substrate for cleavage is a single-stranded RNA (ssRNA) reporting probe. Guided by crRNA, Cas13 identifies the target ssRNA sequence and activates its specific cis-cleavage activity and nonspecific trans-cleavage activity, allowing it to cleave any ssRNA (Shmakov et al., 2015;Zhao et al., 2022). Unlike Cas12, Cas13 relies on a protospacer flanking site (PFS) for target recognition and cleavage, which reduces the base requirements for Cas enzymes in the target sequence (avoiding G bases at the PFS site), expanding the range of detectable target sequences. ...
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) system has proven to be a powerful tool for nucleic acid detection due to its inherent advantages of effective nucleic acid identification and editing capabilities, and is therefore known as the next-generation of molecular diagnostic technology. However, the detection technologies based on CRISPR/Cas systems require preamplification of target analytes; that is, target gene amplification steps through isothermal amplification or PCR before detection to increase target analyte concentrations. This creates a number of testing limitations, such as extended testing time and the need for more sophisticated testing instruments. To overcome the above limitations, various amplification-free assay strategies based on CRISPR/Cas systems have been explored as alternatives, which omit the preamplification step to increase the concentrations of the target analytes. Nanozymes play a pivotal role in enhancing the sensitivity of CRISPR-based detection, enabling visual and rapid CRISPR assays. The utilization of nanozyme exceptional enzyme-like catalytic activity holds great promise for signal amplification in both electrochemical and optical domains, encompassing strategies for electrochemical signal sensors and colorimetric signal sensors. Rather than relying on converting a single detection target analyte into multiple analytes, these methods focus on signal amplification, the main mechanism of which involves the ability to form a large number of reporter molecules or to improve the performance of the sensor. This exploitation of nanozymes for signal amplification results in the heightened sensitivity and accuracy of detection outcomes. In addition to the strategies that improve sensor performance through the application of nanozymes, additional methods are needed to achieve visual signal amplification strategies without preamplification processes. Herein, we review the strategies for improving CRISPR/Cas systems that do not require preamplification, providing a simple, intuitive and preamplification-free CRISPR/Cas system detection platform by improving in-system one-step amplification programs, or enhancing nanozyme-mediated signal amplification strategies.
... However, these tests are inaccurate and have many errors related to false positives and low specificity (27)(28)(29)(30). Therefore, a new rapid assay based on nucleic acid detection is urgently needed to accurately and rapidly detect pathogens, including viruses (31,32). Infectious bronchitis is one of the important respiratory diseases of chickens that cause several economic losses in the Poultry industry. ...
Infectious bronchitis is an acute respiratory disease of poultry associated with reduced egg production and heavy economic losses in chicken flocks. Rabid and accurate detection of IB virus (IBV) is essential for controlling and preventing the infection. In this study, we developed a rapid, accurate, and instrument less assay to detect IBV. For the first time, reverse transcription-Recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas13 (SHERLOCK) was used to rapidly visualize IBV. The novel assay was tested in timing, sensitivity, and specificity. The spike gene (S gene) was used as a target gene for detecting the virus. Three samples were used to optimize the assay; sample form confirmed infected chickens with IB, positive sample (full synthesis of S gene), and negative sample from free IB infected chickens. The results show that the Sherlock-based Cas13 platform is a highly specificity and sensitivity assay for detecting infectious bronchitis virus. The assay detected ten copies per µL of the input RNA. No false positives or cross-reactions were seen when bovine coronavirus (BCV) was used instead of IBV in the tested sample. Readout of the results needs just fifty minutes, including RNA extraction. Furthermore, No instrument was used, and amplification of the virus's nucleic acid was performed at room temperature. Sherlock-based Cas13 should clinically use for rapid diagnosis of infectious bronchitis in chickens. However, further studies and experiments are needed to perform the assay at the sample base without extraction of RNA.
... The forensic science CRISPR-Cas13 version allows it possible to recognize and identify certain RNA sequences, making it easier to diagnose RNA-based infections and analyze gene expression patterns in forensic investigations. [37] Austin Publishing Group ...
Forensic science has significantly evolved with advancements in epigenetics, biotechnology, gene editing and DNA profiling. A major breakthrough in this evolution is the CRISPR-Cas9 technology , introduced in the early 21 st century, it has revolutionized the field, especially enhanced the precision and accuracy of forensic methods and revolutionized genetic research CRISPR-Cas9 greatly enhancing forensic analysis in areas like DNA fingerprinting, kinship testing and forensic phenotyping. It also improved the accuracy of biological evidence analysis, using techniques such as Single Nucleotide Polymorphism (SNP) analysis and forensic epigenetics. As the field advances, it navigates to complexities of legal and ethical implications, ensuring that forensic science continues to progress with integrity and efficacy. This advancement placed forensic science at the forefront of technological and scientific achievement. This progress marks a significant step forward in the realm of scientific investigation and legal justice.
... The manuscript of Zhao L, et al [1] is an excellent review describing the usefulness of the CRISPR-Cas13a system as a tool for molecular diagnostics, gene therapy, gene editing, and RNA imaging. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. ...
This minireview describes some of the articles published in the last two years related to innovative technologies including CRISPR-Cas, surface-enhanced Raman spectroscopy, microfluidics, flow cytometry, Fourier transform infrared spectroscopy, and artificial intelligence and their application to microbiological diagnosis, molecular typing and antimicrobial susceptibility testing. In addition, some articles related to resistance to new antimicrobials (ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol) are also described.
... Fluorescent reporter and quenching groups are labeled at the 5′ and 3′ ends of the non-target RNA sequences, respectively; as the non-target RNA is cleaved, fluorescence is detectable. Therefore, the presence or absence of fluorescence could be used to determine whether Cas13a specifically recognized its target RNA through crRNA (Zhao et al., 2022). ...
Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1–H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/μL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.
