Advancing CRISPR/Cas Biosensing with Integrated Devices

Abstract

Rather than being famous only in the gene editing field, by revealing the collateral cleavage activity of Cas12a, Cas13a, and Cas14 effectors, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) systems (i.e., CRISPR/Cas) have received significant credit in modern analytical science with the capability of detecting versatile analytes with superior sensitivity and specificity. (1,2) A variety of exciting CRISPR/Cas biosensing systems have now been developed successfully for detection of different analytes varying from nucleic acids to non-nucleic acids (such as metabolites, proteins, exosomes, and metal ions). Although the most popular signal output in CRISPR/Cas biosensors is fluorescence, various signal output modalities such as colorimetric, electrochemiluminescence, electrochemical, and electrical have been applied in CRISPR/Cas biosensing systems. Furthermore, the potential of CRISPR/Cas has been demonstrated in multiplex detection by integration with microfluidics or other devices enabling identification of the presence of multiple targets. However, despite extensive efforts and success to develop CRISPR/Cas diagnostic tools based on trans-cleavage enzymatic activity, these systems encounter unavoidable challenges, including inadequate detection limit (near the picomole level) for detecting clinically relevant biomarkers at subpicomolar levels and limited catalytic efficiency for DNA cleavage. These limitations significantly hinder the widespread adoption of CRISPR/Cas diagnostic tools in clinical diagnostics and point-of-care testing. To further enhance detection sensitivity and avoid the necessity for sophisticated and costly equipment, nucleic acids-based preamplification techniques, including thermal-dependent amplification, such as polymerase chain reaction (PCR), and thermal-independent amplification, rolling circle amplification (RCA), recombinase polymerase amplification (RPA), or loop-mediated isothermal amplification (LAMP), are frequently integrated with CRISPR/Cas based assays. Although preamplification techniques significantly increase the sensitivity, they inevitably overshadow Cas effectors and neglect the intrinsic detection capability of Cas effectors. Preamplification also extends detection time and reduces the efficiency of subsequent detection due to nonspecific amplification and primer interference, while substantially increasing the risk of aerosol contamination. The most sensitive nucleic acid amplification strategies employ exponential amplification formats in which amplicons (amplification products) are recycled as primers or templates. However, because of the exponential format, nonspecific background products that lead to false-positive results are inevitable after long reaction times and can be caused by, for example, contaminants, off-template polymerase products, and secondary structures of primers or templates. Therefore, the reaction time of exponential amplification has to be evaluated and controlled in practice to avoid the generation of unwanted signals. Consequently, the sensitivity of exponential amplification-based diagnostics is always determined by the resolution between true- and false-positive signals generated by diluted standard samples and blank/negative controls, respectively. Development of preamplification-free strategies aims to achieve rapid one-pot detection with high detection limit (attomolar or single-molecule detection levels) and high adaptability. This can be achieved by optimizing key components of CRISPR/Cas biosensors (such as reporters), employing cascade amplification techniques, adopting microfluidic droplet analysis, and integrating with other signal readout patterns. Conventional single-strand DNA reporters have been replaced by a range of nanoparticle-based reporters, (3) such as gold nanoparticle (AuNP) reporters, quantum dot reporters, platinum nanoparticles, and aggregation-induced emission agent reporters. AuNPs were used in a CRISPR/Cas12a biosensor integrated with surface enhanced Raman spectroscopy (SERS) signal readout to achieve a detection limit of detection as low as 10 aM. (4) By utilizing autocatalytic nucleic acid circuits, a CRISPR/Cas biosensor enabled DNA detection with the attomolar detection limit without the need for preamplification. (5) Although these systems demonstrated the ability to detect nucleic acids, integration of immunoassays into the CRISPR/Cas system can achieve the detection of other analytes in low abundance. (6) CRISPR/Cas technologies have demonstrated promise in biosensing beyond a simple assay. (7) The integration of CRISPR technology with engineering tools has led to significant advances in molecular biology and healthcare from in vitro diagnosis to in vivo monitoring, from tube assays to integrated devices. (8−10) Integrated with a lateral flow assay, a face mask incorporated with a lyophilized CRISPR/Cas sensor was developed for the noninvasive detection of SARS-CoV-2 at room temperature in 90 min that requires no user intervention other than the press of a button. (11) Microfluidic paper-based analytical devices were also integrated with CRISPR/Cas12a biosensors to realize the supersensitive detection of pathogenic bacteria in foods. (10) A wearable microneedle patch that uses CRISPR-activated graphene biointerfaces was reported for the extraction and long-term monitoring of universal cell-free DNA. It enables the real-time detection of nucleic acid biomarkers over 10 days in vivo, highlighting its potential for early disease screening and prognosis. (12) A single-step CRISPR detection of monkeypox virus in 15 min was developed with a vest-pocket diagnostic device with a sensitivity of 0.5 copies μL^–1 and 100% concordance with real-time PCR in clinical validation, (13) which is adaptable to resource-limited settings. Multiplex detection is challenging due to the collateral cleavage activity of certain Cas enzymes, resulting in interference due to possible cross-reactivity among multiple analytes. (14) Solutions, such as engineering high-fidelity Cas variants, coupling with optimal crRNA designs, using orthogonal Cas effectors, and integrating with microfluidic technologies, allow simultaneous detection of multiple targets without interference. We recently integrated CRISPR-Cas12a immunosensing on glass fiber with a portable fluorescence reader to achieve sensitive detection of various proteins with a low pM detection limit, including cytokines in synovial joint fluids in a point-of-care scenario. (6) By eliminating the expensive instrument and tedious sample preparation, CRISPR/Cas-mediated devices that provide a simple sample-to-answer will continue to bring about a breakthrough in point-of-care diagnosis. (10,11) CRISPR/Cas-based biosensors are much further away from maturation. This editorial encourages submissions on CRISPR/Cas-mediated biosensing devices that demonstrate significant advancements in existing biosensors to address the growing need for quick, cost-effective, sensitive, and accurate field-deployable detection of multiple analytes from in vitro to in vivo. (11) With the integration with nanotechnologies, microfluidics, Cas enzyme-based orthogonal systems, and artificial intelligence, our aim is to achieve preamplification free CRISPR/Cas biosensors to facilitate comprehensive and reliable multiple analyte detection. (15) And it will lead to a breakthrough in high throughput biomarker discovery (16) by designing “all-in-one” devices that combine sample processing, amplification, and readout. Real-time monitoring of target analytes or in vivo bioimaging will be another highlight of the research that CRISPR/Cas biosensors can bring to the biomedical engineering field if novel reporters are discovered to integrate with devices that provide continuous signals corresponding to the target analytes. CRISPR/Cas biosensors typically involve multiple steps, including target extraction, amplification, and signal readout, which can become time-consuming and prone to contamination. In addition to device integration, CRISPR/Cas-mediated detection will respond with innovations in the Cas enzyme and reagent discovery to realize a one-pot assay with desirable detection limit and reduced assay time. Realizing the full potential of these CRISPR/Cas biosensing platforms requires sustained interdisciplinary collaboration between scientists, biologists, engineers, clinicians, and policy makers. Continued innovation in the engineering of CRISPR/Cas components, device integration, and regulatory standardization is essential to translate these cutting-edge technologies from the laboratory to the bedside and beyond, although there are challenges. We are confident that CRISPR/Cas biosensing systems continue to match proudly as diagnostic tools that are not only rapid and accurate but also affordable and globally accessible, driving significant improvements in precision medicine and sustainability in modern analytical science. This article references 16 other publications. This article has not yet been cited by other publications.