Molecular diagnostic technology plays an important role in pathogenic microorganism detection, epidemic prevention and control, disease diagnosis and precision medicine, but it has the disadvantages of long turnaround times, low sensitivity and poor specificity. Therefore, there is an urgent need to develop rapid, sensitive and specific molecular diagnostic techniques. In this study, a single-molecule detection method without amplification was developed by combining the CRISPR/Cas13 system and total internal reflection fluorescence microscopy (TIRF). The conserved 2×HEPN domain of Cas13 protein mutated into dCas13 (deactivated Cas13), resulting in the loss of nuclease activity but retaining the activity of the conjugating enzyme, allowing the dCas13 protein to specifically recognize and bind RNA molecules. Then, the trisomy complex formed by the dCas13 protein, the sgRNA (fluorescent group labeled) and the S gene (target RNA) of the SARS-CoV-2 virus in the reaction system was captured using the capture probe, and the target RNA molecules were detected by TIRF. The experimental results show that, under the condition of no target amplification, the established single-molecule detection method had a detection sensitivity of 1 pmol/L for the target RNA. Compared with the inherent accessory cleavage activity of the CcaCas13b protein, the sensitivity shows a 1000-fold increase. This detection system has high specificity and can effectively distinguish the S gene of the SARS-CoV-2 virus and its common mutants (N501Y and D614G). In addition, the genomic RNA of the SARS-CoV-2 virus (158 ng/µL) was successfully detected using this method. The single-molecule detection technology established in this study affords high sensitivity and strong specificity, and does not require additional nucleic acid amplification steps. Our work provides new ideas for the development of subsequent rapid diagnostic methods and has potential application value.

With the continuous growth of the world population, the demand for high-protein foods such as meat and dairy products is increasing greatly, which brings great challenges to the supply of food proteins in China. Some microbes can utilize carbon dioxide, methane, methanol and other single carbon compounds to produce high-quality single cell proteins (SCP), which can be applied in the food industry. Establishing a green microbial system for the production of SCP is important to ensure the security of the food protein supply in China. In addition, microbial transformation of single carbon compounds to SCP can reduce carbon emissions, alleviate the greenhouse effect, and achieve sustainable development. In this article, we review the application of microbial SCP in the food industry and recent advances in the microbial production of SCP from single carbon compounds, and we describe the metabolic network mechanisms of microorganisms utilizing natural single carbon compounds and speculate on the prospects for the modification of these microorganisms. Finally, we discuss the future prospects for the use of synthetic biology to modify microorganisms to produce SCP from single carbon substrates. With this review, we hope to provide ideas for the commercial production of microbial SCP.

Throughout thousands of years, the yeast Saccharomyces cerevisiae has been acting as a cell factory in food production. Lately, it has further functioned as a platform cell factory for the production of a multitude of different compounds, spanning from high-volume fuels to high-value pharmaceuticals. The past decade has witnessed the fact that the innovative tools in synthetic biology have driven the rapid progress of the yeast cell factory, enabling us to edit the genetic systems of organisms efficiently and "reprogram" elements or systems such as genes, circuits, pathways, and networks of organisms. Here, we will offer a brief review that highlights the most recent significant advances and perspectives regarding the innovative tools in yeast synthetic biology. These tools encompass genome editing tools, computational tools, adaptive laboratory evolution, and the standardization of biological DNA parts, with the intention of providing a practical guide for the implementation of novel, effective, and efficient development of the customized yeast cell factory.