ssDNA Reporter Research Articles

Trans Single‐Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction

AbstractAs a CRISPR‐Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single‐stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1‐based RNA‐activated detection platform (Amplification, Transcription, Cas14a1‐based RNA‐activated trans ssDNA cleavage, ATCas‐RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1‐based ssDNA‐activation. Using ATCas‐RNA via a fluorophore quencher‐labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high‐efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class‐2 Cas systems.

Trans Single-Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction.

As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.

Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.

Exploring the Trans-Cleavage Activity of CRISPR/Cas12a on Gold Nanoparticles for Stable and Sensitive Biosensing

Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.

17454 Leveraging CRISPR-Cas12a for the detection of human T-Cell leukemia virus type 1

Human T-cell leukemia virus type 1 (HTLV-1) is a potent carcinogenic oncovirus that, while asymptomatic in the majority, has the potential to cause devastating complications such as adult T-cell leukemia/lymphoma (ATLL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Despite its high morbidity and global burden, poor access to current testing modalities limits HTLV-1 testing in endemic regions. Here we perform the pre-clinical, proof-of-concept experiments necessary for the development of a sensitive, specific, and low-cost CRISPR-based screening test for HTLV-1. We took advantage of recent reports describing the ability of Cas12a enzymes and guide RNAs (gRNAs) to act as molecular sensors in field-deployable viral diagnostic assays. Upon specific recognition of the gRNA-complementary DNA sequence, eg HTLV-1 proviral DNA, Cas12a/gRNA complexes can catalyze nonspecific trans-cleavage of a single-stranded DNA (ssDNA) reporter, providing an amplified signal of the target recognition event (2). We designed and synthesized multiple HTLV-1-specific Cas12a gRNAs targeted to conserved regions of the HTLV-1 genome. We also designed a custom ssDNA reporter molecule that could be used to measure target-induced Cas12a trans-cleavage activity by quantitative polymerase chain reaction (qPCR). Upon detection of HTLV-1-specific targets in vitro, Cas12/gRNA complexes demonstrated concentration-dependent cleavage of ssDNA. Our assay was able to detect 36 femtomoles of HTLV-1 target DNA and lays the foundation for future efforts to develop a robust, point-of-care CRISPR-based HTLV-1 diagnostic test that could potentially transform the detection and management of HTLV-1 infection.

Cas12a-Activated Universal Field-Deployable Detectors for Bacterial Diagnostics

Field-deployable detectors of disease biomarkers provide a simple and fast analysis of clinical specimens. However, most of the existing field-deployable diagnostics have poor sensitivity and are not suitable for the detection of biomarkers with low abundance. Herein, we report a highly sensitive and rapid colorimetric readout paper-based assay for pathogen detection by integrating the unique collateral activity of a Cas12a-activated universal field-deployable detector (CUFD). The collateral effect of Cas12a results in a nonspecific destruction of a fluorophore biotin-labeled ssDNA reporter for the CUFD. This technique can quantify seven different kinds of pathogens in blood samples without any purification procedure, with sensitivity as low as 10 aM for the Shigella dysenteriae DNA. This CUFD technique has significant potential for the detection of pathogenic DNA as well as other types of DNA or RNA targets at the point-of-care application.

CRISPR-Cas12a based aptasensor for sensitive and selective ATP detection

CRISPR-Cas based detection platform opens up a new avenue for biosensing owing to its programmability, sequence specificity and high base resolution upon nucleic acid targets. In current study, we utilised the target DNA-induced non-specific single-stranded DNA cutting activity (trans-cleavage) of CRISPR-Cas12a to fabricate an aptamer mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamer as the target ssDNA for Cas12a, thereby in the presence of ATP, the ATP-binding aptamer would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration dependent change in fluorescence resulted from trans-cleavage of doubly labeled ssDNA reporter. In this way, we successfully “translated” ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 μM to 200 μM and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecule by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.

An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids

CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with CRISPR/Cas and loop-mediated Isothermal Amplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5α pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.

CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease

ABSTRACT CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.