Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection.

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.

Sequential Therapy of Acute Kidney Injury with a DNA Nanodevice.

The high demand for acute kidney injury (AKI) therapy calls the development of multifunctional nanomedicine for renal management with programmable pharmacokinetics. Here, we developed a renal-accumulating DNA nanodevice with exclusive kidney retention for longitudinal protection of AKI in different stages in a renal ischemia-reperfusion (I/R) model. Due to the prolonged kidney retention time (>12 h), the ROS-sensitive nucleic acids of the nanodevice could effectively alleviate oxidative stress by scavenging ROS in stage I, and then the anticomplement component 5a (aC5a) aptamer loaded nanodevice could sequentially suppress the inflammatory responses by blocking C5a in stage II, which is directly related to the cytokine storm. This sequential therapy provides durable and pathogenic treatment of kidney dysfunction based on successive pathophysiological events induced by I/R, which holds great promise for renal management and the suppression of the cytokine storm in more broad settings including COVID-19.

COVID-19: A Call for Physical Scientists and Engineers.

The COVID-19 pandemic is one of those global challenges that transcends territorial, political, ideological, religious, cultural, and certainly academic boundaries. Public health and healthcare workers are at the frontline, working to contain and to mitigate the spread of this disease. Although intervening biological and immunological responses against viral infection may seem far from the physical sciences and engineering that typically work with inanimate objects, there actually is much that can-and should-be done to help in this global crisis. In this Perspective, we convert the basics of infectious respiratory diseases and viruses into physical sciences and engineering intuitions, and through this exercise, we present examples of questions, hypotheses, and research needs identified based on clinicians' experiences. We hope researchers in the physical sciences and engineering will proactively study these challenges, develop new hypotheses, define new research areas, and work with biological researchers, healthcare, and public health professionals to create user-centered solutions and to inform the general public, so that we can better address the many challenges associated with the transmission and spread of infectious respiratory diseases.

SARS-CoV-2 RNA reference materials

Coronavirus disease (COVID-19) is an acute infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Reverse transcription real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was the firstly authorized method for the detection of SARS-CoV-2 RNA. As this method is sensitive, specific, it has been widely recognized as the golden standard for the diagnosis of COVID-19. Unfortunately, several false-negative cases have been reported after the outbreak of COVID-19, probably due to the quality of the kits or the improper operation of RT-qPCR. Nucleic acid reference materials (RM) are the key element for the metrology traceability and quality control of SARS-CoV-2 RNA detection, but the development of RNA RM remains a challenge in the biology metrology field. Two main problems are the low stability of the RNA sample and the lack of proven absolute quantification methods. To establish the measurement traceability for SARS-CoV-2 RNA detection, a novel RNA reference material (RM) was developed. The RM is a mixed solution of 3 in vitro transcribed RNA molecules which cover different key target sequences of SARS-CoV-2 gene: The full-length of nucleoprotein (N) gene (28274-29533, GenBank: MT027064.1), the full-length of envelope protein (E) gene (26245-26472, GenBank: MT027064.1), and partial sequence of open reading frame 1ab (ORF1ab) (13321-15540, GenBank: MT027064.1). The purity of the transcribed RNA molecules was verified by a biological analyzer. The results showed that the molecular length of all the RNA molecules were consistent with our design. The clear peaks of our RNA RMs strongly demonstrated good purity. For absolute quantification of RNA RMs, we studied digital PCR (dPCR) for RNA samples. Digital PCR evenly partitioned the sample and PCR reaction solution to a very large number of units, on a microporous chip or in the liquid droplets, etc. After a PCR amplification reaction, the fluorescence signal was detected for each unit individually, with a binary readout of "0" or "1" for negative and positive results respectively. Through the statistics of positive results based on the Poisson distribution, the copy number of RNA sample was accurately determined without standard curves needed. Digital PCR has significantly higher reliability and accuracy. Mainly based on the PCR primers and probes for SARS-CoV-2 detection suggested by the Chinese CDC and WHO, we optimized the key factors of dPCR towards improved amplification efficiency. Through digital PCR measurements by 4 laboratories, the certified values of concentration (copies/mu L) were assigned for the N gene, E gene, and ORF1ab gene in the mixed RM. Single-stranded RNA is unstable and easy to be degraded by RNase in the environment, thus the optimization of RNA protectants is very important for the stability of RNA RMs. During the study of the stability, we found that a proper protector (1 mmol/L DTT and 0.5 U/L Rnase Inhibitor) can effectively increase the valid storage life of our RNA RM. Based on the latest data, the concentration of our RNA RMs was stable for at least 30 d under -80 degrees C storage and 13 d under -4 degrees C storage. In order to verify the applicability of our RNA RM in the actual virus detection process, we analyzed our RMs using 9 SARS-CoV-2 nucleic acid detection kits. These virus RNA detection kits were from different manufacturers with various detection principles, that are being applied in laboratories for virus detection. Finally, our RNA RMs showed high generalizability among 9 kits. The development of RNA RM provides the metrological basis for the quality control of SARS-CoV-2 detection kits.