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Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology. [ABSTRACT FROM AUTHOR]

The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full‐size RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on both single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA‐Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double‐stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe nosocomial threat, prompting a need for efficient detection methods. Traditional approaches, such as bacterial culture and PCR, are time-consuming and cumbersome. The CRISPR-based gene editing system offered a potential approach for point-of-care testing of CRAB. Methods: We integrated recombinase polymerase amplification (RPA) and CRISPR-Cas12a system to swiftly diagnose CRAB-associated genes, OXA51 and OXA-23. This multiplex RPA-CRISPR-Cas12a system eliminates bulky instruments, ensuring a simplified UV lamp-based outcome interpretation. Results: Operating at 37°C to 40°C, the entire process achieves CRAB diagnosis within 90 minutes. Detection limits for OXA-51 and OXA-23 genes are 1.3 × 10−6 ng/μL, exhibiting exclusive CRAB detection without cross-reactivity to common pathogens. Notably, the platform shows 100% concordance with PCR when testing 30 clinical Acinetobacter baumannii strains. Conclusion: In conclusion, our multiplex RPA coupled with the CRISPR-Cas12a system provides a fast and sensitive CRAB detection method, overcoming limitations of traditional approaches and holding promise for efficient point-ofcare testing. [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudorabies virus (PRV) is the causative pathogen of Aujeszky’s disease. It causes high mortality and miscarriage rates in the infected swine, leading to tremendous economic losses in the global swine industry. PRV has also been demonstrated to trigger viral encephalitis in humans. The eradication policy and large-scale vaccination have been adopted globally as the most effective strategy against PRV. A simple, fast, and sensitive diagnostic method is highly demanded to differentiate between vaccinated and infected swine. Herein, we designed a detection system combining multienzyme isothermal rapid amplification (MIRA) and CRISPR-Cas12a (termed MIRA-Cas12a), characterized by high sensitivity and specificity, low cost, less equipment, and convenient visualization. By targeting the gB, gE, and TK genes of PRV, the MIRA-Cas12a assay is able to distinguish the infected, uninfected, and vaccinated swine by the naked eyes in 40 min (from DNA extraction to result readout) and with comparable sensitivity to conventional quantitative PCR. A 37°C heater and a source of blue light are all the equipment required to detect PRV. Thus, the MIRA-Cas12a detection will facilitate PRV surveillance and minimize the financial losses to the swine industry. IMPORTANCE Pseudorabies virus (PRV) causes high mortality and miscarriage rates in the infected swine, and the eradication policy coupled with large-scale vaccination of live attenuated vaccines has been adopted globally against PRV. Differential diagnosis of the vaccinated and infected swine is highly demanded. Our multienzyme isothermal rapid amplification (MIRA)-Cas12a detection method described in this study can diagnose PRV with a superior sensitivity comparable to the quantitative PCR (qPCR) and a competitive detection speed (only half the time as qPCR needs). The portable feature and the simple procedure of MIRA-Cas12a make it easier to deploy for clinical diagnosis, even in resource-limited settings. The MIRA-Cas12a method would provide immediate and accurate diagnostic information for policymakers to respond promptly.

Summary: Existing CRISPR/Cas12a‐based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans‐cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans‐cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a‐RRVRR variant presented robust trans‐cleavage activity and looser PAM constraints. Moreover, we found the existing one‐pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single‐base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a‐RRVRR variant‐mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR‐based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light‐start CRISPR‐Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one‐pot assay is achieved. The photocontrolled one‐pot assay is simpler and is 50‐fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10–20 min is sufficient for effective detection, which is much faster than the current gold‐standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety. [ABSTRACT FROM AUTHOR]

Rapid, accurate and visual point-of-care testing (POCT) methods for pathogenic bacteria detection are essential for avoiding foodborne diseases caused by pathogens or their toxins. In this study, we proposed a rapid and visual detection method that we named "Cas12aVIP". By combining recombinase polymerase amplification (RPA), a CRISPR/Cas12a system and a cationic-conjugated polythiophene derivative (poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT) mixed with single-stranded DNA (ssDNA)), the solution turned red in the absence of the target DNA based on conformational modifications of the conjugated backbone of PMNT, whereas it displayed yellow, thus realizing the colorimetric detection of DNA. The Cas12aVIP method yielded high specificity and no interference from other nontargeted bacteria. The detection was accomplished in 40 min and the signal could be observed by the naked eye under natural light, presenting great potential for a variety of rapid nucleic acid detection applications without requiring technical expertise or ancillary equipment. [ABSTRACT FROM AUTHOR]

To establish an invasive aquatic species detection method with high sensitivity and specificity, we used the CRISPR/Cas12a system. After comparing the mitochondrial COX1, COX2, COX3, and CYTB sequences of many invasive species, we designed and synthesized guide RNA (gRNA) and amplified target sequences for a detection system based on binding of the Cas12a protein. We then used alligator gar target DNA as a standard to assess the detection system. The reaction system was optimized in terms of Cas12a and gRNA concentrations, ssDNA reporter concentration, and reaction temperature, and this optimized system was used to detect other invasive species. The results showed that 100 nmol/L Cas12a, 100 nmol/L gRNA, and 700 nmol/L ssDNA reporter added to 20 μL of a DNA sample at 42 °C was optimal, with the system able to detect 10.3 fM target DNA of an invasive species with good specificity and sensitivity. The results were directly observed under UV light irradiation. Moreover, we applied eDNA (environmental DNA), which can trigger the generation of fluorescent signals in the system, and the denaturation-annealing-extension cycles of PCR were optimized so that the detection system works when the cycle number is as low as 25 and even when the water samples from the species habitat are diluted 10−24-fold. Our findings indicate that the established method is effective for the detection of invasive aquatic species. • Engineered Crispr/Cas12a system successfully detect target DNA of an invasive species. • The detection system was sensitive enough to detect target DNA of an invasive species at a minimum of 10.3 fM. • The detection system is highly specific. [ABSTRACT FROM AUTHOR]

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions. [ABSTRACT FROM AUTHOR]

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 100 copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20–30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks. [ABSTRACT FROM AUTHOR]

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A novel thermodynamic approach for integrating isothermal amplification with CRISPR detection in a closed assay vessel without the use of interior driving forces or human intervention was developed by harnessing internal air thermal expansion for autonomous liquid actuation by coupling a thermally responsive microfluidic cartridge with a simple two-step temperature profile. [Display omitted] • Seamless integration of CRISPR detection and target preamplification. • Automated actuation without external driving mechanism or human intervention. • Temperature-responsive architecture utilizes the internal air thermal expansion. • It shortens the sample-to-answer time without compromising sensitivity. The rising incidence of syphilis highlights the urgent need to develop molecular tests of Treponema pallidum at the point-of-care (POC). Traditional molecular approaches based on polymerase chain reaction (PCR) often involve expensive equipment, expert technicians, and lengthy turnaround time. Recently emerged approaches coupling CRISPR detection with isothermal amplification techniques hold great promise to advance the POC diagnostics, but commonly require multiple manual operations for postamplification reaction transfer or suffer from compromised performance in terms of sensitivity or assay time. To address these issues, we develop a thermometer-inspired microfluidic biosensing platform that seamlessly integrates CRISPR detection and isothermal amplification through temperature-programmed air expansion for intended fluidic manipulation. This platform not only allows for automated running of the canonical two-step recombinase polymerase amplification-CRISPR/Cas12a assay via a simple two-step temperature profile, but also provides a smartphone-enabled fluorescence readout with a limit of detection of a few DNA copies in 30 min. We detail the rational design of a thermally-responsive microfluidic cartridge and showcase how it harnesses the temperature difference in preamplification and CRISPR detection to implement the assay workflow without the need for an external driving mechanism or human intervention. Furthermore, we validate its applicability for rapid and sensitive detection of Treponema pallidum DNA in clinical samples (n = 30), exhibiting 100 % sensitivity and 100 % specificity compared to the quantitative PCR technique. The simplicity and effectiveness may lead this platform to be a powerful upgrade kit compatible with the existing CRISPR approaches, thus facilitating their widespread point-of-care diagnostic applications. [ABSTRACT FROM AUTHOR]

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional onepot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed. [ABSTRACT FROM AUTHOR]

Lumpy skin disease (LSD) is a severe and highly infectious pox disease of cattle caused by the lumpy skin disease virus (LSDV). To facilitate early control of LSD, this study aimed to develop a new rapid on-site LSDV detection method using an orf068 gene-based recombinase polymerase amplification assay (RPA) coupled with a CRISPR-Cas12a-based fluorescence assay (RPA-Cas12a-fluorescence assay). The results showed that the sensitivity of our RPA-Cas12a-fluorescence assay for detecting LSDV orf068 gene reached 5 copies/μL with plasmid as a template, and 102 TCID50/mL with viral genomic DNA as a template. No cross-reaction with other common bovine viruses was observed. Further, an on-site RPA-Cas12a-fluorescence assay of 40 clinical samples from cattle with or without LSD showed a diagnostic sensitivity of 96.3% (95% CI: 81.0–99.9%) and specificity of 92.31% (95% CI: 62.1–99.6%), which was close to those of the quantitative PCR assay. Therefore, our RPA-Cas12a-fluorescence assay has promising prospects in on-site rapid LSDV detection. [ABSTRACT FROM AUTHOR]

Desirable biosensing assays need to be sensitive, specific, cost‐effective, instrument‐free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal‐ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR‐Cas12a, crRNA and cauliflower‐like large‐sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD‐bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS‐CoV‐2 genomic RNA and adenosine. We also engineer a cell phone app and a hand‐held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point‐of‐care diagnostics. [ABSTRACT FROM AUTHOR]

Abstract Background As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. Results Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. Conclusions In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

Rapid, accurate and visual point-of-care testing (POCT) methods for pathogenic bacteria detection are essential for avoiding foodborne diseases caused by pathogens or their toxins. In this study, we proposed a rapid and visual detection method that we named “Cas12aVIP”. By combining recombinase polymerase amplification (RPA), a CRISPR/Cas12a system and a cationic-conjugated polythiophene derivative (poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT) mixed with single-stranded DNA (ssDNA)), the solution turned red in the absence of the target DNA based on conformational modifications of the conjugated backbone of PMNT, whereas it displayed yellow, thus realizing the colorimetric detection of DNA. The Cas12aVIP method yielded high specificity and no interference from other nontargeted bacteria. The detection was accomplished in 40 min and the signal could be observed by the naked eye under natural light, presenting great potential for a variety of rapid nucleic acid detection applications without requiring technical expertise or ancillary equipment.

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 100 copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20–30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks.

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis - and trans -cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing. [Display omitted] • Engineered crRNAs with variable stem-loop designs demonstrate adaptability with Cas12a • cCRISPR broadens the gene editing toolbox by screening 207 combinations of Cas12a and crRNAs • cCRISPR detects SARS-CoV-2 in saliva, nasopharyngeal swabs, and tracheal aspirates Nguyen et al. explore the diversity and adaptability of 23 Cas12a orthologs in combination with their canonical and 8 noncanonical crRNAs. A total of 207 combinations of Cas12a and crRNAs were applied for clinical nucleic acid detection and gene editing in cells. The data expand the toolbox for next-generation CRISPR-based diagnostics and genome engineering. [ABSTRACT FROM AUTHOR]

Developing rapid and accurate pathogen detection methods is increasingly important, and CRISPR-Cas system can be optimized for this purpose. CRISPR-Cas12a is a single RNA-guided endonuclease system with the potential for nucleic acid detection. There is a broad diversity among Cas12a nucleases with robust detection capability. Herein, we characterised three Cas12a orthologs (ObCas12a, MbCas12a, and ScCas12a), including cis - and trans -cleavage activities, the identification of PAM, single-base resolution ability, and the application for nucleic acid detection. These Cas12a orthologs displayed robust cis - and trans -cleavage activities, and performed well in terms of specificity and sensitivity for nucleic acid detection. Furthermore, they have subtle differences in single-base resolution and recognised PAM sites in vitro. Therefore, these Cas12a nucleases are candidate proteins for CRISPR-based diagnostic methods. Addition of these enzymes to the nucleic acid detection toolbox will further expand the utility of this powerful technology. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics. • CRISPR-Cas12a trans-cleaves G-triplex structures. • G-triplex formed by TBA11 is a promising Cas12a-based biosensing reporter. • TBA11 reporter enhances the detection sensitivity up to 20 folds over normal ssDNA reporter. • The LOD of our assay achieved 0.1 aM for the amplified HPV16 plasmid. • A specificity and sensitivity of 100% and 94.7% was achieved in the detection of HPV patient samples. [ABSTRACT FROM AUTHOR]