Leveraging Cas13a's trans-cleavage on RNA G-quadruplexes for amplification-free RNA detection.

In this study, we investigated Cas13a's efficacy in trans-cleaving RNA G-quadruplexes (rG4s) as an alternative to ssRNA reporters in CRISPR-Cas13a diagnostics. Our findings demonstrate enhanced efficiency due to the structural arrangement of rG4s. Implementing a simplified CRISPR-Cas13a system based on rG4, we identified SARS-CoV-2 infections in 25 patient samples within 1 hour without target pre-amplification.

Integrating CRISPR-Cas12a into a Microfluidic Dual-Droplet Device Enables Simultaneous Detection of HPV16 and HPV18.

Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10-18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.

Coupling CRISPR/Cas12a and Recombinase Polymerase Amplification on a Stand-Alone Microfluidics Platform for Fast and Parallel Nucleic Acid Detection.

Timely identification of human papillomavirus (HPV) infection is crucial for the prevention of cervical cancer. Current HPV detection methods mainly rely on polymerase chain reaction (PCR), which often requires bulky equipment and a long assay time. In this work, we report a heating-membrane-assisted multiplexed microfluidics platform that couples recombinase polymerase amplification (RPA) and CRISPR technology (termed M3-CRISPR) for fast and low-cost detection of multiple HPV subtypes. The heating membrane can provide convenient temperature control for the on-chip RPA and CRISPR assays. This stand-alone system allows simultaneous detection of HPV16 and HPV18 with high specificity and detection sensitivity (0.5 nM and 1 × 10-18 M for unamplified and amplified plasmids, respectively) in 30 min with a fluorescence-based readout. Furthermore, we introduced an optimized lateral flow dipstick (LFD) into the portable system to allow visualized detection of HPV DNA. The LFD-based readout also reached a detection sensitivity of 1 × 10-18 M for amplified plasmids and realized successful detection of HPV subtypes in the clinical samples. Finally, we established an automatic microfluidic system that enables the sample-in-answer-out detection of HPV subtypes. We believe that this fast, convenient, and affordable molecular diagnostic platform can serve as a useful tool in point-of-care testing of HPV or other pathogens.

Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping.

Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence- and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.

Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification.

Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a microfluidic device with CRISPR-Cas12a and multiplex recombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection.

CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.

Optical design and fabrication of an all-aluminum unobscured two-mirror freeform imaging telescope.

Freeform optics offers more degrees of freedom to optical design that can benefit from a compact package size and a large field of view for imaging systems. Motivated by the advances in modern optical fabrication and metrology, freeform optics has been found in many applications. In this paper, we will describe the challenging optical design, fabrication, metrology, and assembly of an all-aluminum unobscured two-mirror freeform imaging telescope. The telescope has a large field of view of 20∘×15∘. The freeform aluminum mirrors are manufactured by diamond turning based on a feedback modification strategy. The freeform mirrors are measured by a computer-generated hologram-based interferometric null test method. All-aluminum configuration has the advantages of being athermal and cost-effective.

A highly selective and sensitive near-infrared fluorescent probe for imaging of hydrogen sulphide in living cells and mice.

Hydrogen sulphide (H2S), the third endogenous gaseous signalling molecule, has attracted attention in biochemical research. The selective detection of H2S in living systems is essential for studying its functions. Fluorescence detection methods have become useful tools to explore the physiological roles of H2S because of their real-time and non-destructive characteristics. Herein we report a near-infrared fluorescent probe, NIR-HS, capable of tracking H2S in living organisms. With high sensitivity, good selectivity and low cytotoxicity, NIR-HS was able to recognize both the exogenous and endogenous H2S in living cells. More importantly, it realized the visualization of endogenous H2S generated in cells overexpressing cystathionine ß-synthase (CBS), one of the enzymes responsible for producing endogenous H2S. The probe was also successfully applied to detect both the exogenous and endogenous H2S in living mice. The superior sensing properties of the probe render it a valuable research tool in the H2S-related medical research.