Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

Application of the CRISPR/Cas System in Pathogen Detection: A Review.

Early and rapid diagnosis of pathogens is important for the prevention and control of epidemic disease. The polymerase chain reaction (PCR) technique requires expensive instrument control, a special test site, complex solution treatment steps and professional operation, which can limit its application in practice. The pathogen detection method based on the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated protein (CRISPR/Cas) system is characterized by strong specificity, high sensitivity and convenience for detection, which is more suitable for practical applications. This article first reviews the CRISPR/Cas system, and then introduces the application of the two types of systems represented by Type II (cas9), Type V (cas12a, cas12b, cas14a) and Type VI (cas13a) in pathogen detection. Finally, challenges and prospects are proposed.

Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay.

To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/µl in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, glyceraldehyde-3-phosphate dehydrogenase was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by reverse transcription polymerase chain reaction (RT-PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector.

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus.

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

Commentary - CRISPR-based techniques: Cas9, Cas13 and their applications in the era of COVID-19.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system enables scientists to edit diverse genome types with relative ease, with the aim - in the near future - to prevent future human beings from developing genetic diseases. The new opportunities arising from the system are broad-ranging and revolutionary, but such prospects have also been the cause for alarm throughout the international scientific community. The authors have laid out a review of the trials carried out so far in terms of genome editing, for the ultimate purpose of weighing implications and criticisms. We feel that possible valuable alternatives, such as induced pluripotent stem cells should not be overlooked.

Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2.

The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.

Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.

CRISPR-based detection of SARS-CoV-2: A review from sample to result.

The current pandemic of the 2019 novel coronavirus (COVID-19) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concern. Rapid, affordable, and accurate diagnostics of SARS-CoV-2 is essential for early treatment and control of the disease spread. In the past few years, CRISPR technology has shown great potential for highly sensitive and specific molecular diagnostics. Amid the ongoing COVID-19 pandemic, there is an increasing interest in implementing CRISPR-based diagnostic principles to develop fast and precise methods for detecting SARS-CoV-2. In this work, we reviewed and summarized these CRISPR-based diagnostic systems as well as their characteristics and challenges. We also provided future perspectives of CRISPR-based sensing towards point-of-care molecular diagnosis applications.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.