Search

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3′-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases. • Cas12a crRNAs induce DNA cleavage in lysate of human cells expressing AsCas12a. • Designed crRNAs are localized in mitochondria of transfected human cells. • crRNAs mitochondrial targeting doesn't require fusion with import determinants. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a p adlock activator which could i nhibit the t rans -cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca2+ dependent. In the presence of Ca2+, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans -cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg2+, Mn2+, Cd2+, Cu2+, Na+, Al3+, K+, Fe2+, and Fe3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSD s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system. [Display omitted] [ABSTRACT FROM AUTHOR]

CRISPR/Cas technology is a versatile tool for genome engineering in many organisms, including filamentous fungi. Cpf1 is a multi-domain protein of class 2 (type V) RNA-guided CRISPR/Cas endonuclease, and is an alternative platform with distinct features when compared to Cas9. However, application of this technology in filamentous fungi is limited. Here, we present a single CRISPR/Cpf1 plasmid system in Aspergillus aculeatus strain TBRC 277, an industrially relevant cell factory. We first evaluated the functionality of three Cpf1 orthologs from Acidaminococcus sp. BV3L6 (AsCpf1), Francisella tularensis subsp. novicida U112 (FnCpf1), and Lachnospiraceae bacterium (LbCpf1), in RNA-guided site-specific DNA cleavage at the pksP locus. FnCpf1 showed the highest editing efficiency (93 %) among the three Cpf1s. It was further investigated for its ability to delete a 1.7 kb and a 0.5 kb from pksP and pyr G genes, respectively, using two protospacers targeting these gene loci in a single crRNA array. Lastly, simultaneous editing of three sites within TBRC 277 genome was performed using three guide sequences targeting these two genes as well as an additional gene, kusA , which resulted in combined editing efficiency of 40 %. The editing of the NHEJ pathway by targeting kusA to generate a NHEJ-deficient strain of A. aculeatus TBRC 277 improved gene targeting efficiency and yielded more precise gene-editing than that of using wild-type strain. This promising genome-editing system can be used for strain improvement in industrial applications such as production of valuable bioproducts. [ABSTRACT FROM AUTHOR]

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus posing significant economic challenges in shrimp aquaculture. Depending on the shrimp species, IHHNV infections can lead to mass mortalities, deformities, or growth retardation, all of which impact production output. Timely identification of IHHNV, particularly using nucleic acid detection methods, is paramount for effective prevention. However, PCR and methods relying on nucleic acid probes face challenges due to the presence of chromosome-integrated, endogenous viral elements (EVEs) of IHHNV sharing identical sequences to their infectious counterparts. While long-range PCR assays, such as LongAmp PCR (LA-PCR), have shown promise in minimizing false positive results, their applicability in the field is limited by their long turnaround times and reliance on costly equipment. To address these limitations, we have developed a rapid and potentially field-deployable platform termed C as9- RPA - C as12a ("CRPAC") capable of differentiating the infectious form of IHHNV from EVEs, capitalizing on the fact that the genome of IHHNV is single-stranded, while EVEs are double-stranded DNA. This method employs Cas9 endonuclease to deplete EVEs, thereby enriching for single-stranded genomic DNA. This enriched DNA is then subjected to recombinase polymerase amplification (RPA) coupled with the Cas12a diagnostic assay. Here, we show that CRPAC gives positive results exclusively for infectious IHHNV, appearing as strong fluorescence or a distinct pattern on lateral flow strips that facilitates naked-eye evaluation. In field sample testing, CRPAC demonstrated excellent agreement with LA-PCR, identifying 8 out of 16 samples containing infectious IHHNV, while the WOAH-recommended PCR assays gave false positive results to all samples containing EVEs. Collectively, our findings underscore the utility of CRPAC as a robust and field-adaptable tool for distinguishing between infectious IHHNV and EVEs, irrespective of their nucleotide sequence similarities. [Display omitted] • CRPAC combines recombinase polymerase amplification with dual CRISPR systems. • CRPAC is free from false positives arising from endogenous viral elements of IHHNV. • CRPAC allows naked-eye visualization through fluorescent and lateral flow readouts. • Faster than PCR and not requiring thermal cycling, CRPAC suits field applications. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual's genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy. • Split G4 was utilized as the label-free output method for Cas12a system. • Cascade strand displacement reaction rendered the platform great specificity. • The detection sensitivity reached 1 copy per test with the help of RPA. • Human buccal swab samples were tested with high accuracy. • The platform could serve as a universal platform for SNP discrimination. [ABSTRACT FROM AUTHOR]

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N 6 - methyladenosine (m6A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m6A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m6A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m6A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m6A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m6A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2–3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m6A fractions in real biological samples, even in low abundance RNA biomarkers. [Display omitted] • DCAS can directly determine the presence of m6A and its fractions in single-base resolution. • The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy. • This method can be successfully employed to accurately determine m6A fractions in low abundance RNA biomarkers. [ABSTRACT FROM AUTHOR]

APE1, an essential enzyme for DNA repair, is overexpressed in various cancers and has been identified as a potential biomarker for cancer diagnosis. However, detecting APE1 at low expression levels in the early stage of cancer presents a significant obstacle. Here, we introduced a novel localized Cas12a-based cascade amplification (LCas12a-CA) method. This method confined both the terminal deoxynucleotidyl transferase and the crRNA/Cas12a complex onto the surfaces of gold nanoparticles (AuNPs). This confinement not only boosts the stability of the multiple enzymes but also induces a substrate channeling effect. As a result, it significantly accelerates the reaction rate and enhances the sensitivity of APE1 detection. Upon the addition of APE1, the AP sites within the APE1 primer can be recognized and cleaved by APE1, exposing the 3′-OH ends. In the presence of LCas12a-CA, polyA sequences are generated at 3′-OH ends with the help of TdT and dATP. The sequences directly enter the Cas12a system, activating the trans -cleavage activity of Cas12a, thereby cutting the reporters on the surface of AuNPs and releasing fluorescence. Our platform demonstrates a detection limit (LOD) as low as 2.51 × 10−6 U/mL, which is more than 60 times lower than that of free Cas12a-CA. Furthermore, the LCas12a-CA exhibits enhanced resistance ability in extreme environments and has been proven effective for the detection of APE1 in clinical samples. Overall, this work offers a promising platform for robust biosensing in cancer diagnosis and prognosis. [Display omitted] • LCas12a-CA generated a substrate channeling-like effect, enabling one-pot and sensitive detection of APE1 with a LOD of 2.51 × 10−6 U/mL. • LCas12a-CA exhibits excellent stability, maintaining enzyme activity in extreme conditions. • LCas12a-CA has strong anti-interference capability, enabling sensitve APE1 detection in complex samples，making it suitable for clinical cancer diagnosis. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation. [ABSTRACT FROM AUTHOR]

• Bacteria inhibit assembly between magnetic covalent organic frameworks and ssDNA. • The hybridization chain reaction and CRISPR/Cas12a realize signal amplification. • As low as 10 CFU/mL of total bacteria can be quantified within 50 min. The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans -cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects. [ABSTRACT FROM AUTHOR]

Nucleic acids are essential biomarkers in molecular diagnostics. The CRISPR/Cas system has been widely used for nucleic acid detection. Moreover, canonical CRISPR/Cas12a based biosensors can specifically recognize and cleave target DNA, as well as single-strand DNA serving as reporter probe, which have become a super star in recent years in the field of nucleic acid detection due to its high specificity, universal programmability and simple operation. However, canonical CRISPR/Cas12a based biosensors are hard to meet the requirements of higher sensitivity, higher specificity, higher efficiency, larger target scope, easier operation, multiplexing, low cost and diversified signal reading. Then, advanced non-canonical CRISPR/Cas12a based biosensors emerge. In this review, applications of non-canonical CRISPR/Cas12a-based biosensors in nucleic acid detection are summarized. And the principles, peculiarities, performances and perspectives of these non-canonical CRISPR/Cas12a based biosensors are also discussed. [Display omitted] • Brief overview of canonical/non-canonical CRISPR/Cas12a biosensors. • Characterization and mechanism of non-canonical CRISPR/Cas12a biosensors. • Advance and perspective of CRISPR/Cas12a biosensors for nucleic acid detection. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a is a promising tool for nucleic acid detection. However, due to the protein flexibility, Cas12a tolerates mismatches, which limits its specificity. In this study, the single-molecule assay revealed that the length of crRNA regulates the association kinetics between crRNA/Cas12a complex and target DNA. Short-crRNA/Cas12 associates the target dsDNA 2-fold faster than the single-nucleotide mismatched dsDNA, whereas the long-crRNA/Cas12a binds to the fully matched and single-nucleotide mismatched DNA targets with similar rates. These findings are further corroborated by electrophoretic mobility shift assay (EMSA) and double-stranded DNA (dsDNA) cleavage results. Inspired by these findings, we established stem-loop amplification conjugated short crRNA CRISPR-Cas12a (SlashCas12a) detection. In this approach, stem-loop-mediated microRNA reverse transcription was harnessed to enhance the amplification efficiency of the short single-stranded RNA. The short-crRNA Cas12a specifically distinguishes the single mismatched miRNA homologs, i.e. let-7a family members. The introduction of PAM containing stem-loop can break through the limitation of PAM for Cas12a and efficiently amplify the miRNAs. Multiple miRNAs, including miR-122b, miR-21, and let-7a, can be efficiently detected, and the limit of detection is up to 7.8 aM. Furthermore, the distinct expression of miR-21 and let-7a can be detected in the lung adenocarcinoma and breast samples using this method. These results demonstrate the appropriate crRNA engineering will extend the application of Cas12a in the specific molecule diagnosis. • The single-molecule assay reveals the crRNA recognition mechanism and guides the rational design of crRNA. • Stem-loop amplification conjugated short crRNA CRISPR-Cas12a (SlashCas12a) breaks through the limitation of PAM for Cas12a. • The SlashCas12a detects up to 7.8 aM target sequence and distinguishes the single mismatch among the let-7 family members. [ABSTRACT FROM AUTHOR]

The enzymatic actions of endonucleases in vivo can be altered due to bound substrates and differences in local environments, including enzyme concentration, pH, salinity, ionic strength, and temperature. Thus, accurate estimation of enzymatic reactions in vivo using matrix-dependent methods in solution can be challenging. Here, we report a matrix-insensitive magnetic biosensing platform that enables the measurement of endonuclease activity under different conditions with varying pH, salinity, ionic strength, and temperature. Using biosensor arrays and orthogonal pairs of oligonucleotides, we quantitatively characterized the enzymatic activity of EcoRI under different buffer conditions and in the presence of inhibitors. To mimic a more physiological environment, we monitored the sequence-dependent star activity of EcoRI under unconventional conditions. Furthermore, enzymatic activity was measured in cell culture media, saliva, and serum. Last, we estimated the effective cleavage rates of Cas12a on anchored single-strand DNAs using this platform, which more closely resembles in vivo settings. This platform will facilitate precise characterization of restriction and Cas endonucleases under various conditions. [ABSTRACT FROM AUTHOR]

Fusarium verticillioides is an important phytopathogenic fungus that poses a threat to maize yield and quality in global maize-growing regions by causing Fusarium ear and stalk rot. The fungus is known to produce fumonisins, which are toxic secondary metabolites and have been associated with high incidences of esophageal cancer. The FUM1 gene is responsible for producing a crucial polyketide synthase required for fumonisin biosynthesis and is present in all pathogenic strains of F. verticillioides. This study aims to develop a rapid and accurate detection assay for F. verticillioides based on the FUM1 gene, by utilizing Chelex-100 resin for DNA extraction, recombinase polymerase amplification coupled with CRISPR/Cas12a cleavage and lateral flow detection (RPA-Cas12a-LFD) assay. The developed RPA-Cas12a-LFD assay exhibited remarkable specificity for F. verticillioides , and the lowest limit of detection was 2 ag DNA of F. verticillioides. The entire diagnostic process was completed in just 73 min, including sample DNA extraction, RPA reaction, Cas12a cleavage, and result readout. Furthermore, RPA-Cas12a-LFD assay was found to be equivalent to single-spore isolation and partial translation elongation factor 1α gene (TEF-1α) sequencing in identifying diseased samples in the field. In summary, this accurate and portable detection equipment has great potential for detecting and recognizing F. verticillioides , especially in areas where sophisticated lab equipment is not available. • The developed RPA-Cas12a-LFD assay exhibited extremely high specificity for toxigenic F. verticillioides. • The developed RPA-Cas12a-LFD assay detected as low as 2 ag DNA of F. verticillioides. • The developed RPA-Cas12a-LFD assay was validated by testing field samples without sophisticated laboratory equipment. [ABSTRACT FROM AUTHOR]

CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems. Engineering CRISPR nucleases, such as Cas9 and Cas12a, has proven to be a good strategy for improving CRISPR technology as a genome engineering toolset. Introducing exogenous protein domains can generate new functions in CRISPR/Cas systems, including highly specific genome editing, base editing, gene expression regulation, and genome imaging. Deletion of certain protein domains can generate smaller CRISPR nucleases with reduced activity. Engineering specific protein domains in Cas9 or Cas12a can result in altered protospacer-adjacent motif (PAM) specificity and on-target specificity for improved CRISPR/Cas performance. [ABSTRACT FROM AUTHOR]

Genome-editing CRISPR-Cas systems, using Cas9 and Cas12a endonucleases, have improved our ability to precisely edit genomes and control gene expression. We summarize here the knowledge gained from using CRISPR-Cas9 and CRISPR-Cas12a in fungal research. Also discussed are strategies developed for limiting the occurrences of off-target mutations caused by CRISPR-Cas genome editing. [ABSTRACT FROM AUTHOR]

Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labeled around the PAM sites of no target sequence(NTS), and antibody would bind on the labeled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labeled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets. [ABSTRACT FROM AUTHOR]

Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. This assay combines the merits of high affinity of aptamer, trans -cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications. [Display omitted] • A CRISPR/Cas12a linked aptamer sandwich assay for thrombin is developed. • Either aptamer or antibody is used as capture element on microplate. • Active DNA of Cas12a is connected with detection aptamer for signal amplification. • The assay can detect thrombin as low as 10 pM without pre-amplification. • The assay is selective for thrombin and can work in complex biological samples. [ABSTRACT FROM AUTHOR]

Streptococcus suis serotype 2 is an economically important zoonotic pathogen that causes septicemia, arthritis, and meningitis in pigs and humans. S. suis serotype 2 is responsible for substantial economic losses to the swine industry and poses a serious threat to public health, and accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a high-fidelity detection and serotyping platform for S. suis serotype 2 based on recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system called Cards-SSJ/K. Cards-SSJ had a detection limit of 10 CFU, takes <60 min, and no cross-reaction was found with other S. suis serotypes, closely related Streptococcus spp., or common pig pathogens, and Cards-SSK could differentiate serotype 2 from serotype 1/2. Results from Cards-SSJ and qPCR were equivalent in detecting S. suis serotype 2 in tissue samples. Analysis indicated that despite a relatively high reagent cost compared to PCR and qPCR, Cards-SSJ was less time-consuming and had low requirements for equipment and personnel. Thus, it is an excellent method for point-of-care detection for S. suis serotype 2. [Display omitted] • Cas12a/crRNA assisted rapid detection and serotyping of Streptococcus suis serotype 2 and 1/2. • Fast identification without the need for expensive instruments in 1 h • Versatile readout styles (naked eye, fluorescence) can be chosen. • Suitable for use in the field and also diagnostic laboratories. [ABSTRACT FROM AUTHOR]

In this study, we developed a novel electrochemical biosensor based on CRISPR/Cas12a (E-CRISPR) for the rapid and sensitive detection of Salmonella Typhimurium (S. Typhimurium). The CRISPR/Cas12a system was applied to identify S. Typhimurium gene and induce signal changes in electrochemical measurement. The colloidal gold and MXene (CG@MXene) nanocomposites were synthesized and immobilized to improve the performance of the biosensor by decreasing the background noise. The formation process of CG@MXene was well characterized, and experiment conditions were fully optimized. Under the optimal conditions, the proposed E-CRISPR biosensor exhibited excellent sensitivity for S. Typhimurium , with a limit of detection (LOD) of 160 CFU/mL, and great specificity against other common foodborne pathogens. Furthermore, the feasibility of the E-CRISPR biosensor was evaluated by analyzing S. Typhimurium -spiked chicken samples, with a recovery rate ranging from 100.46% to 106.37%. In summary, this research proposed a novel E-CRISPR biosensor from a new perspective to detect S. Typhimurium which can be an alternative approach for bacterial detection in the food supply chain. The scheme of the proposed E-CRISPR biosensor for S. Typhimurium detection, which mainly consists of three parts: electrode modification, CRISPR/Cas12a-mediated nucleic acid target recognition, CRISPR/Cas12a-induced signal output. [Display omitted] • The CRISPR/Cas12a system, serveded as signal recognition and amplification system, provides benefits for sensitive detection. • The CG@MXene nanocomposites were utilized to reduce the background signal of the electrode. • The biosensor demonstrated the ability to detect S. Typhimurium at 160 CFU/mL with a simple sensing workflow. • The biosensor showedits potential for application in food safety testing. [ABSTRACT FROM AUTHOR]

• Production of Ad vectors was suppressed by expression of Cas12a. • Regulation of Cas12a expression by Tet-on system or conditional promoter improved the titer of Ad vectors. • Suppression of Cas12a expression in packaging cells improved the titer of Ad vectors. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches. [ABSTRACT FROM AUTHOR]

The yellow head virus complex comprises 8 genotypes of single-stranded RNA viruses infecting penaeid shrimp, but only yellow head virus genotype 1 (YHV1) is capable of causing massive shrimp mortalities and severe economic losses. However, current molecular techniques could not detect YHV1 with high specificity, or require complicated protocols for this purpose. Here, we developed a combination of reverse-transcriptase recombinase polymerase amplification (RT-RPA) and CRISPR-Cas detection ("RR-Cas") for the genotype-specific detection of YHV1. The method can be completed within 1.5 h at a constant temperature near 37 °C, after which the results can be visualized by the naked eye using fluorescent, lateral flow, and G-quadruplex DNAzyme-assisted colorimetric assays. The detection limit is 100 copies of plasmid DNA and 100 fg of total RNA from YHV1 infected shrimp, comparable to the WOAH-recommended one-step RT-PCR protocol. In the evaluation of clinical samples, RR-Cas was found to be less sensitive than nested RT-PCR, but displayed superior specificity, yielding no false positive results towards samples containing YHV2 and 3. Overall, our findings suggest that RR-Cas is a powerful addition to the toolkit for genotype-specific viral diagnosis in shrimp aquaculture, with the potential to greatly streamline disease surveillance and management in the shrimp industry. [Display omitted] • RR-Cas specifically detects YHV1 and does not cross react with YHV2–8. • The method is isothermal, fast, and detects 100 fg YHV1-infected RNA at the minimum. • Fluorescent, lateral flow, and DNAzyme assays can be used for naked-eye detection. • Integrating G-quadruplex DNAzyme enables low-cost, colorimetric visualization. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas proteins have been employed as innovative tools for biomolecule detection. However, the functionality of the Cas protein is limited by the RNA-guided nuclease that cleaves pre-amplified products to generate fluorescence signals. As such, we proposed a strategy to use the Cas12a protein for active primer production and as an initiator for subsequent isothermal amplification. Target nucleic acids activate Cas12a to cleave the DNA component of RNA-DNA chimera primers with a 3′ phosphate group, converting inactive RNA-DNA chimera primers into active ones. We then used two isothermal amplification methods based on Klenow DNA polymerase/T7 RNA polymerase and terminal deoxynucleotidyl transferase to generate fluorescence and colorimetric signals, respectively. The Salmonella enterotoxin (stn) gene was effectively amplified with satisfactory sensitivity and selectivity even in real samples such as drinking water and human serum, paving the way for Cas12a-derived active primers that may be useful in diverse detection platforms. [Display omitted] • We devised a strategy to utilize Cas12a for the production of active primers used for subsequent isothermal amplification. • The versatile utility of the active primer was demonstrated via two isothermal amplification methods. • The fluorescence and colorimetric detection systems were constructed. • This method determined the stn gene with satisfactory sensitivity and specificity. [ABSTRACT FROM AUTHOR]

The white spot syndrome virus (WSSV) is a pathogenic virus that has caused serious problems in aquaculture. An effective therapy for its infection is not yet available, and it is essential to develop a fast-response diagnosis method. This study established an RPA and CRISPR-Cas12a combined one-pot method for WSSV detection. The one-pot method was achieved by rational design and systematic optimization of the reaction, including concentrations of Cas12a and crRNA, and the buffer condition. The sensitivity reached 101 copies/reaction, which was in the highly sensitive range when compared to all the available molecular detection assays. Moreover, the specificity and field sample validation results were satisfactory. The method has integrated the potent amplification capacity of RPA and the precise cleavage-activation property of CRISPR-Cas12a into one simultaneous reaction, thus has enhanced convenience and less risk of carry-over contaminations. The method is conducted under 37 °C and only demands for minimum laboratorial support. It provides a convenient and reliable on-site diagnosis tool for WSSV infection in aquaculture. • A one-pot RPA-CRISPR detection method for WSSV is established. • The method reaches a high sensitivity of 101 copies/reaction. • The method is suitable for on-site diagnosis of WSSV infection. [ABSTRACT FROM AUTHOR]

Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This highlights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3′ end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR + CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies μL-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1 h). Because each method has its own set of advantages, the laboratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future. [Display omitted] • Both CRISPR-Cas12a-based approaches effectively distinguished single-base alterations. • RPA introduced mismatched bases into the adjacent sites of mutated bases. • To prevent PCR amplifications of the non-target, single-base mutations were added to the 3′ end of the primers. [ABSTRACT FROM AUTHOR]

Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV–Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 102 copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis. [Display omitted] • Cas12a and universal AuNPs strand-displacement probe for SARS-CoV-2 assay. • The oligo trigger acts as switch for signal change and substrate of Cas12a. • The color change can be visual with the naked eye and UV–Vis spectrometer. • A low-cost hand warmer is used as an incubator for visual assay within 20 min [ABSTRACT FROM AUTHOR]

The exceptional programable trans -cleavage ability of type V and VI CRISPR/Cas nucleases paved the way for ultrasensitive CRISPR/Cas based sensing of nucleic acid and alternative targets. However, the enhancement of the trans -cleavage activity of Cas effector with organic chemical agents has not been explored thus far. We report here chemically enhanced trans -cleavage activity of Cas12a and Cas13a nucleases which improves sensor performance in CRISPR/Cas biosensing. Improved trans -ssDNA cleavage of Cas12a and trans -ssRNA cleavage of Cas13a were demonstrated by using sulfhydryl reductants and non-ionic surfactants. DTT and PVA were demonstrated to be the most effective chemical enhancers in both cases. By using a fluorescence resonance energy transfer (FRET)-based intramolecular distance measurements, we identified the mechanism of this enhancement to be the conformation change of the ribonucleoprotein and quantified it to be major (about 50% increase of a relevant intramolecular distance). These chemical enhancers have been integrated into the established CRISPR/Cas biosensing protocols without additional modifications. For the detection of Helicobacter Pylori DNA and SARS-CoV-2 RNA, we found a decreased reaction time by 75–83% and 4–6-fold increased sensitivity. These results indicate that chemical enhancers provide a versatile and broadly applicable approach to break through the barriers of long reaction time and sensitivity in CRISPR/Cas sensors. [Display omitted] • Enhanced trans -cleavage activity of Cas12a and Cas13a by chemical enhancers. • Sulfhydryl reductants and non-ionic surfactants are two types of chemical enhancers. • The mechanism of this enhancement is the conformation change of Cas RNP. • The reaction time of chemical enhanced CRISPR/Cas system reduced 75–83%. • The sensitivity of chemical enhanced CRISPR/Cas system increased 4–6 folds. [ABSTRACT FROM AUTHOR]

Pathogen nucleic acid detection is of great significance to control the spread of diseases caused by the viruses. Nevertheless, traditional methods for nucleic acid detection such as polymerase chain reaction (PCR) and oligonucleotide microarrays require bulky instruments, which restrain their point-of-care (POC) testing application. Here, we proposed a POC method enabling sensitive detection of pathogen nucleic acids by combining the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a-based assay and personal glucometer readout (PGM). The quantification of target pathogen DNA by PGM was achieved based on pathogen DNA activates Cas12a ssDNase to cleave magnetic bead-DNA-invertase reporter probe, and separated free invertase to catalyze hydrolysis of sucrose to glucose. Without using nucleic acid amplification technology, we demonstrated here dual signal amplifications based on Cas12a and invertase-mediated catalytic reactions, making it possible to sensitively detect HIV-related DNA or SARS-CoV-2 pseudovirus with the limits of detection of 11.0 fM and 50 copies/μL, respectively. This strategy also showed excellent selectivity as well as potential applicability for detection of HIV in human serum samples or of SARS-CoV-2 in saliva samples. Therefore, our CRISPR-PGM-based dual signal amplifications detection platform might offer a great promise in POC diagnosis of pathogen nucleic acids. We developed an improved CRISPR-PGM method without extra nucleic acid amplification process for sensitive POC detection of pathogen nucleic acids. In this sensing platform, two successive signal amplification steps were fully taken advantaged. One step is target DNA-activated Cas12a nuclease to release invertase from magnetic beads by trans -cleavage reaction. The second is the separated invertase catalyzes hydrolysis of sucrose to glucose at temperature with highly catalytic efficiency (50 °C). The presented CRISPR-PGM system might offer a great promise in POC detection of HIV or of SARS-CoV-2 in combination with reverse transcriptase reaction. [Display omitted] • A dual amplified CRISPR-PGM platform was proposed for pathogen nucleic acids test. • The method is convenient, rapid, and economical, and meets the needs of POC assay. • The method was used to sensitively detect HIV-related DNA or SARS-CoV-2 N gene. [ABSTRACT FROM AUTHOR]

On-site and real-time clinical monitoring have been progressed dramatically by integrating biosensor science with portable digital electronic technology. Clustered regularly interspaced short palindromic repeats (CRISPR) with association of RNA-guided nucleases (CrRNA-Cas enzymes) have achieved novel CRISPR/Cas biosensing science as a promising revolutionized diagnostic technology for portable and on-site healthcare monitoring and diagnostics. Among several available CRISPR/Cas systems, CRISPR/Cas12a and CRISPR/Cas13a conjugates are utilized broadly in biosensor design, because of their capability to cleave both target and non-target sequences. With the advantages of portability, cost-effectiveness, facile operation, high durability, and reproducibility, CRISPR/Cas-based biosensing techniques are a perfect choice for designing ultra-sensitive point-of-care diagnostic devices with amplified response signals. In the present review, we summarize the advances in the CRISPR/Cas-based biosensors with the focus on healthcare and diagnostic purposes. The cooperation of nanomaterial engineering with CRISPR/Cas biosensors is also represented to attain a promising viewpoint for offering novel user-friendly test kits for announcing ultra-low levels of diverse targets in the future. • CRISPR/Cas biosensing science is a promising diagnostic technology for portable healthcare monitoring and diagnostics. • CRISPR/Cas12a and CRISPR/Cas13a conjugates are utilized broadly in biosensor design. • Herein, we summarize the advances in the CRISPR/Cas based biosensors with the focus on healthcare and diagnostic purposes. • The cooperation of nanomaterial engineering with CRISPR/Cas based biosensors is also represented. • This article represents a promising viewpoint for offering novel user-friendly test kits for announcing ultra-low levels of diverse targets in the future. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease. [ABSTRACT FROM AUTHOR]

A novel ECL biosensor was developed for ultrasensitive mercury ions (Hg2+) determination based on gold nanoparticles-coated nitrogen-doped carbon dots (Au@CNDs) as luminophores and exonuclease III (Exo III) as well as CRISPR/Cas12a as signal amplifiers. The biosensor was fabricated by pre-modifying Au@CNDs hybrids and ferrocene-labeled single-stranded DNA (Fc-ssDNA) layer in sequence on the interface of a gold working electrode. The initial Au@CNDs ECL of the biosensor is quenched by Fc. Then, a thymine-rich ssDNA probe was designed for the recognition of Hg2+. In the presence of Hg2+, this thymine-rich ssDNA probe was folded to a DNA duplex based on T-Hg2+-T mismatches. Then, Exo III catalyzes the digestion of duplex of hairpin DNA, liberating short single-stranded DNA (ssDNA) product and releasing Hg2+. The produced ssDNA can be recognized by CRISPR/Cas12a and further trigger the collateral cleavage ability of CRISPR system, which can indiscriminately cut short Fc-ssDNA on the electrode, producing an ECL signal of Au@CNDs. Under the optimized conditions, a detection limit of 0.45 fM was realized for Hg2+ determination. The efficiency and robustness of this biosensor were further verified by using environmental water and human serum samples, which demonstrated its potential application in the environment and biological science. • A novel ECL biosensor for ultrasensitive Hg2+ determination was proposed. • The biosensor combined exonuclease III with Cas12a to achieve duplex signal amplification. • The biosensor realized ultrasensitive Hg2+ detection at sub-femtomolar level. • The work provided a new avenue for other toxic metal ions determination. [ABSTRACT FROM AUTHOR]

Recent advances in CRISPR/Cas biosensing have led to impressive performance in sensitivity, specificity, and speed for nucleic acid detection. However, the remarkable advantages (such as universality, ultralow, attomolar detection limits) of CRISPR/Cas biosensing systems are limited in testing non-nucleic acid targets. Herein, by synthesizing a functional hybrid conjugate of antibody and single strand DNA oligonucleotide, we had successfully demonstrated the capability to integrate CRISPR/Cas12a-based signal amplification into different types of immunoassay schemes without the need for any additional recognition molecule or molecular synthesis during the detection process, thus providing a simple but generally applicable approach to improve the conventional immunoassays with attomolar sensitivity for small protein detections, referred as the CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). CRUISE is capable of being integrated into various immunoassays either through the primary antibody or the secondary antibody, with sensitivity down to 1 fg mL−1 (∼50 aM) and 6 logs of linear range for detecting cytokines, such as IFN-γ and EGFR, under 3–4 h. It has a 103 times higher sensitivity compared to a commercial IFN-γ ELISA kit, but uses the same experimental scheme. The same 1 fg mL−1 sensitivity along with 6 logs of linear range was realized for IFN-γ detection in human plasma samples. We are expecting that our CRUISE provides an alternative but simple, user-friendly and effective strategy for those who rely on the use of immunoassays, while struggling with the limits of their sensitivity or detection ranges. [Display omitted] • A universal and user-friendly approach to integrate CRISPR/Cas12a trans -cleavage into mainstream immunoassay schemes. • No need for additional target recognition molecules except antibody. • Same experimental protocols and setup as conventional immunoassays. • Breaks through the sensitivity barrier of conventional sandwich immunoassay, such as ELISA. • Capable of reaching aM-level sensitivity for small protein diagnostics but with shorter time. [ABSTRACT FROM AUTHOR]

CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system. • A method for one-step microRNA detection based on CRISPR-Cas12a-assisted catalytic hairpin assembly (CHA) was developed. • The RNase and DNase activities of Cas12a were fully exploited. • It integrated the inherently high sensitivity and specificity both from CHA and CRISPR-Cas12a system. • This method was able to detect target miRNA down to femtomolar concentration. [ABSTRACT FROM AUTHOR]

In addition to its application in genome editing, the CRISPR/Cas system provides a new molecular tool for the construction of biosensor platforms due to its high target sequence specificity and programmability. To exploit the potential of the CRISPR/Cas system for detecting non-nucleic-acid targets, two ultrasensitive biosensors (sensor-ss and sensor-ds) consisting of allosteric aptamer probes integrated with the CRISPR/Cas12a system were designed and constructed for tobramycin detection. Both sensors utilized hairpin DNA containing a tobramycin aptamer sequence as the target recognition probe, which can respond to target binding and further produce signal transduction sequences. However, there are two differences between the sensors. First, in sensor-ss, single-stranded DNA was set to be the activator to trigger cutting activity (trans -cleavage) of CRISPR/Cas12a, leading to cleavage of the FQ-reporter (a short single-stranded DNA labeled with a fluorophore and quencher at each end) and increasing the fluorescent signal. However, double-stranded DNA containing a protospacer adjacent motif (PAM) site was produced in sensor-ds as the signal transduction sequence, which was recognized by CRISPR/Cas12a, resulting in the cleavage of the FQ-reporter. Second, sensor-ss involved a strand displacement amplification (SDA) reaction as the signal enhancement step, while no DNA amplification procedure was designed in sensor-ds. Through DNA sequence and reaction condition optimization, sensor-ds showed a linear relationship between the fluorescence response and tobramycin concentrations ranging from 10 to 300 pM with a limit of detection (LOD) as low as 3.719 pM. Although sensor-ss exhibited higher sensitivity (LOD = 1.542 pM) than sensor-ds, rapidly reaching the fluorescent signal saturation platform led to a narrow linear response range (5–30 pM). Moreover, our CRISPR/Cas12a-based assay can realize on-site detection by the naked eye readout under UV light. The constructed sensor-ds was also used to detect tobramycin in milk and lake water samples, and the results indicated that the detection system was reliable. The design strategy reported in this study could shed new light on CRISPR/Cas12a-based biosensor construction for small molecule detection. • Two biosensors consisting of the allosteric aptamer probes and CRISPR/Cas12a system were designed for tobramycin detection. • The two sensors utilized single- and double-stranded DNA to trigger the cleavage activity of CRISPR/Cas12a, respectively. • The performance of the two sensors were investigated and compared. • The biosensors can realize highly sensitive (LOD = 1.452–3.719 pM), specific and visual determination of trace tobramycin. [ABSTRACT FROM AUTHOR]

Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL−1 (58.8 aM). It also has a tuneable linear detection range that can be easily adjusted within the range 1 fg mL−1 to 100 pg mL−1 (5 orders of magnitude), meeting the requirements of the demanding diagnostic scenarios. CAFI has successfully been demonstrated by detecting IFN-γ from a diverse complex biological sample type, including human serum, whole blood, perspiration, and saliva. Moreover, CAFI is applicable for the detection of other analytes by simply modifying the capture antibody and detection aptamer, demonstrated here with insulin. All these superior capabilities of CAFI make it a suitable technology to measure proteins in low (100 μL) volume complex biological samples. [Display omitted] • CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) for the detection of small proteins. • Detection aptamer triggered CRISPR/Cas12a biosensing system for signal amplification. • CAFI has 5 orders of tuneable detection range with 1 fg mL−1 sensitivity. • CAFI is capable of detection of targets from complex biological samples. [ABSTRACT FROM AUTHOR]

The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans -cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a–MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection. [Display omitted] • A Cas12a based methylation sensing method was developed. • The methylation percentage of a single CpG methylation site was identified and quantified. • The effect of short target lengths on Cas12a′s trans cleavage was quantified. • The effect of cleavage position is determined when combining restriction enzymes and Cas12a. [ABSTRACT FROM AUTHOR]

• A CRISPR/Cas12a-based recipe for a broadly applicable ELISA sensitivity amplification. • Capable of integrated into commercial HRP-based ELISA kit without changing the original reagents or setup. • A possible solution to rapidly deliver a user-friendly and affordable sensitivity amplifier to end-users. • A hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. • 2 orders of magnitude increase in sensitivity of a commercial HRP-based ELISA kit for IFN-γ detection. The collateral cleavage function is only a corollary of programmable nuclease activity of certain Cas effectors, such as Cas12 and Cas13, but it can be utilised to amplify fluorescence signals in various CRISPR/Cas-based biosensing systems. In this work, this special signal amplification capability of CRISPR/Cas12a ribonucleoproteins has been employed to increase the sensitivity of a broad class of commercial ELISA systems with undisclosed chemistry except for the use of horseradish peroxidase (HRP), a common signal reporting molecule. We demonstrated that such ELISA systems with HRP on the detection antibody, can be amplified by 2 orders of magnitude using an example commercial IFN-γ ELISA kit where the detection limit was decreased from 312.5 pg/mL to 1.2 pg/mL. The detection range was simultaneously increased from 2 orders to 3 orders of magnitude. Our CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. The CES-Amplifier can be directly integrated into commercial ELISA kits to replace their original last step, without any additional changes of the ELISA kit reagents or setup. In this way, our CES-Amplifier provides a versatile and affordable approach for expanding CRISPR/Cas-based biosensing to a wide range of non-nucleic acid analytes. [ABSTRACT FROM AUTHOR]

• Integrated CRISPR with spherical nucleic acid for HIV/HPV DNAs multiplex detection. • Asignal-switchable electrochemiluminescence biosensor using a single luminophores. • Sensitive and nucleic acids amplification-free method. • Successful development and validation with spiked human serum sample. Herein, we report a "on-off" signal-switchable electrochemiluminescence (ECL) biosensor for simple, sensitive and quantitative detection of two kinds of virus genes, human immunodeficiency virus (HIV) and human papilloma virus (HPV-16) DNAs, by the integration of spherical nucleic acid (SNA) with CRISPR/Cas12a. The concept behind this approach is "on" signal based on a sandwich type assay after ECL luminophores of carbon dots (CDs) labeled SNA incubated with the biosensor in the presence of HIV DNA; and "off" signal upon the biosensor incubation with assistant DNA (HIV DNA sequence), CDs-labeled SNA and Cas12a/crRNA/HPV-16 DNA ternary complex relying on the collaterally cleavage of Cas12a. The cross-interference between HIV and HPV-16 DNAs can be avoided by adding Cas12a effector to the system or not. Using this strategy, HIV and HPV-16 DNAs are successfully quantitatively detection with detection limits of 30 fM and 0.32 pM (S/N = 3), respectively. Importantly, this system does not rely on multiplex signal reporting molecules, thus simplifying the detection system and avoiding the likelihood of cross interferences presented in the assay. Furthermore, multiplex detection of HIV/HPV-16 DNAs in human serum samples are successfully achieved within 2 h without additional nucleic acid amplification step. As demonstrated, the proposed biosensor has the potential for multiplex detection in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (E-CRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high-sensitivity, and high-accuracy platform for L. monocytogenes detection. • Application of CRISPR/Cas12a system for electrochemical sensing. • The proposed electrochemical transduction strategy provides a way to improve the sensitivity of CRISPR/Cas12a-based methods. • The integrated biosensor enables highly-specific detection of Listeria monocytogenes. • The calculated LODs of L. monocytogenes are 0.68 aM genomic DNA and 940 cfu/g in spike sample within 2 h, respectively. • The biosensor shows excellent practical performance in natural food samples. [ABSTRACT FROM AUTHOR]

CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies. • Cas12a-based electrochemiluminescence biosensor was first proposed. • L-Methionine stabilized gold nanoclusters (Met-AuNCs) was served as high-efficiency ECL emitters. • A detection limit of 0.48 pM for HPV-16 DNA was achieved without any amplification methods. • The Cas12a-based ECL biosensor exhibited excellent practical performance in undiluted human blood samples. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) are the key proteins for the decision of cell fates, and they have been recognized as potent markers for diagnostic and treatment of diseases. Herein, we report on a highly sensitive biosensor for the detection of TFs based on the CRISPR/Cas12a system. This biosensor was accomplished based on the competitive binding of the Cas12a-crRNA and TFs towards a dsDNA referred to as activator. Without TFs, the activator can be recognized by Cas12a-crRNA and cause the activation of the DNase activity of Cas12a. When TFs were added, the TFs can bind with the activator because the activator was designed to contain the specific binding sites of target TFs. We find that this binding can inhibit the association between Cas12a-crRNA and the activator, which hinders the activation of Cas12a. As a proof-of-concept, the rapid detection of five kinds of TFs was presented, and the detection was extended to the analysis of TFs expression in xenograft solid tumors from mice. This investigation is the first attempt to apply CRISPR technology in the sensing of TFs, and it discloses that the blocking of activator can be applied as a new sensing mechanism for the development of CRISPR-based biosensor. • It is found that TFs can compete and inhibit the dsDNA-induced activation of Cas12a. • CRISPR technology is first applied in the biosensing of transcription factors. • The inhibition-based sensing mechanism for CRISPR is proposed. • Rapid sensing of multiple TFs with picomolar sensitivity is realized. [ABSTRACT FROM AUTHOR]

Summary Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing. Graphical Abstract Highlights • Phosphorothioate-modified DNA oligonucleotides inhibit Cpf1-mediated genome editing • Inhibition of Cpf1 activity is time and dose dependent and target sequence independent • psDNA oligonucleotides may block the complex formation of Cpf1-crRNA-DNA • psDNA oligonucleotides inhibit Cas9-mediated genome editing Li et al. show that phosphorothioate-modified DNA (psDNA) oligonucleotides inhibit Cpf1-mediated genome-editing activity in a sequence-independent manner in human cells. These psDNA oligonucleotides interact with Cpf1 protein and block the formation of Cpf1-crRNA-target DNA complex. They also display inhibitory effects on the CRISPR-Cas9 system. [ABSTRACT FROM AUTHOR]