The era of Cas12 and Cas13 CRISPR-based disease diagnosis.

Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems, since their discovery, have found growing applications in cell imaging, transcription modulation, therapeutics and diagnostics. Discovery of Cas12 and Cas13 have brought a new dimension to the field of disease diagnosis. These endonucleases have been extensively used for diagnosis of viral diseases in humans and animals and to a lesser extent in plants. The exigency of SARS-CoV-2 pandemic has highlighted the potential of CRISPR-Cas systems and sparked the development of innovative point-of-care diagnostic technologies. Rapid adaptation of CRISPR-chemistry combined with sensitive read-outs for emerging pathogens make them ideal candidates for detection and management of diseases in future. CRISPR-based approaches have been recruited for the challenging task of cancer detection and prognosis. It stands to reason that the field of CRISPR-Cas-based diagnosis is likely to expand with Cas12 and Cas13 playing a pivotal role. Here we focus exclusively on Cas12- and Cas13-based molecular diagnosis in humans, animals and plants including the detection of SARS-coronavirus. The CRISPR-based diagnosis of plant and animal diseases have not found adequate mention in previous reviews. We discuss various advancements, the potential shortfalls and challenges in the widespread adaptation of this technology for disease diagnosis.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022

CriSNPr, a single interface for the curated and de novo design of gRNAs for CRISPR diagnostics using diverse Cas systems.

CRISPR-based diagnostics (CRISPRDx) have improved clinical decision-making, especially during the COVID-19 pandemic, by detecting nucleic acids and identifying variants. This has been accelerated by the discovery of new and engineered CRISPR effectors, which have expanded the portfolio of diagnostic applications to include a broad range of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline necessitates customized detection schemes based on the fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is especially relevant for variant detection, a low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPRDx currently exists. In this manuscript, we fill this lacuna using a unified web server, CriSNPr (CRISPR-based SNP recognition), which provides the user with the opportunity to de novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. Furthermore, we provide a database of curated pre-designed gRNAs as well as target/off-target for all human and SARS-CoV-2 variants reported thus far. CriSNPr has been validated on multiple Cas proteins, demonstrating its broad and immediate applicability across multiple detection platforms. CriSNPr can be found at http://crisnpr.igib.res.in/.

AN INTEGRATED RT-LAMP AND CRISPR ASSAY FOR NUCLEIC ACID DETECTION IN A SINGLE MICROFLUIDIC CHIP

We will present a microfluidic assay to detect SARS-CoV-2 RNA from nasopharyngeal swab samples. Our method leverages isotachophoresis (ITP) to integrate sample preparation, RT-LAMP, and CRISPR-based nucleic acid detection in an automatable chip. For the first time, we use ITP to purify, pre-concentrate and isothermally amplify target nucleic acids into a ~1 µL reaction volume on-chip. The device then transitions LAMP amplicons into an on-chip zone containing Cas12-gRNA complexes and reporter molecules to measure target-activated CRISPR activity. We will use our method to automatically detect COVID-19 from nasopharyngeal swab samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

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.

Synthetic Biology-Powered Biosensors Based on CRISPR/Cas Mediated Cascade Signal Amplification for Precise RNA Detection

Synthetic biology enabling technologies have been harnessed to create new diagnostic technologies. However, most strategies involve error-prone amplification steps and limitations of accuracy in RNA detection. Here, a cell-free synthetic biology-powered biosensing strategy, termed as SHARK (Synthetic Enzyme Shift RNA Signal Amplifier Related Cas13a Knockdown Reaction), could efficiently and accurately amplify RNA signal by leveraging the collateral cleavage of activated Cas13a to regulate cell-free enzyme synthesis. Based on cascade amplification and tailored enzyme output, SHARK behaves broad compatibility in different scenarios. The portable device based on SHARK was successfully used as SARS-CoV-2 biosensors with high sensitivity and selectivity, and the results were highly consistent with Ct values of qRT-PCR. In addition, when combined with machine learning, SHARK performs bio-computations and thus for cancer diagnosis and staging based on 64 clinical samples. SHARK shows characteristics of precise recognition, cascade amplification and tailored signal outputting comparisons with established assays, presenting significant potential in developing next-generation RNA detection technology.

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.

CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.

The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications.

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection.

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

CRISPR-based assays for rapid detection of SARS-CoV-2.

COVID-19 pandemic posed an unprecedented threat to global public health and economies. There is no effective treatment of the disease, hence, scaling up testing for rapid diagnosis of SARS-CoV-2 infected patients and quarantine them from healthy individuals is one the best strategies to curb the pandemic. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid based tests are considered to be more sensitive with respect to serological tests but present gold standard qRT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. CRISPR based novel techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here, we discuss and present compilation of state-of-the-art detection techniques for COVID-19 using CRISPR technology which has tremendous potential to transform diagnostics and epidemiology.

Trends and Innovations in Biosensors for COVID-19 Mass Testing.

Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID-19. This work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID-19 pandemic has overwhelmed testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral-flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral-flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.