CRISPR-Cas13a system: A novel tool for molecular diagnostics

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system

Functional characterization of the virulence determinant ESX-1 from Mycobacterium tuberculosis

Tuberculosis (TB) is a chronic infectious disease that mainly affects the lungs and causes extensive human morbidity and mortality. It results from infection with Mycobacterium tuberculosis, a slow-growing intracellular pathogen that can replicate and survive inside macrophages. M. tuberculosis relies on the specialised ESX-1 secretion system to export virulence factors needed for intracellular spread and pathogenesis. The ESX-1 apparatus is a multi-subunit nanomachine composed of ~20 polypeptides including membrane proteins, ATPases, proteases, chaperones and substrates. Although many of the individual components of this secretion system have been characterised, the overall mechanism underlying ESX-1 secretion is still far from clear. To obtain a more comprehensive picture of the functioning of the ESX-1 apparatus, we have studied various components which were largely unexplored in M. tuberculosis. The structural component EccE1, the ESX-1 specific protein EspL and the transcriptional regulator WhiB6 have been investigated in this thesis using an integrative approach involving genetics, biochemistry, proteomics and microscopy. We have demonstrated that EccE1 is a membrane- and cell-wall associated protein critical for secretion of ESX-1 substrates and M. tuberculosis-mediated cell lysis. Deletion of eccE1 from the chromosome severely compromised secretion of EsxA, EsxB, EspA and EspC but not EspB. Localization studies using a florescent-fusion protein showed that EccE1 localises to the poles of M. tuberculosis in the presence of an active ESX-1 system. Our study also shows that EspL is a cytosolic protein needed for stabilising EspE, EspF and EspH, suggesting that it acts as a specific chaperone of the ESX-1 secretion system. Moreover, EspL was shown to interact with EspD and to be important for the secretion of ESX-1 substrates. Lack of EspL resulted in a growth defect ex vivo, loss of cytotoxicity and reduction of innate cytokine production demonstrating its critical role in M. tuberculosis virulence. Analysis of the transcriptional response revealed that the only gene deregulated in the absence of espL was whiB6, encoding a transcriptional factor that positively controls ESX-1 genes. To explore the role of this regulator in M. tuberculosis virulence, we generated a deletion mutant of whiB6 and discovered that its loss resulted in severe reduction of cytotoxicity ex vivo. Overall this investigation improves our current understanding of the ESX-1 secretion system and of the molecular basis of M. tuberculosis virulence. The increased knowledge of the complex interactions between the pathogen and the human host will hopefully translate into new strategies to control the spread of TB

Electrochemical control of reversible DNA hybridisation : for future use in nucleic acid amplification

Denaturation and renaturation is indispensable for the biological function of nucleic acids in many cellular processes, such as for example transcription for the synthesis of RNA and DNA replication during cell division. However, the reversible hybridisation of complementary nucleic acids is equally crucial in nearly all molecular biology technologies, ranging from nucleic acid amplification technologies, such as the polymerase chain reaction, and DNA biosensors to next generation sequencing. For nucleic acid amplification technologies, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most commonly used method, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. This Thesis explored the possibility of using electrochemical means to control reversible DNA hybridisation by using electroactive intercalators. First, fluorescence-based melting curve analysis was employed to gain an in depth understanding of the reversible process of DNA hybridisation. Fundamental properties, such as stability of the double helix, were investigated by studying the effect of common denaturing agents, such as formamide and urea, pH and monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The intercalators investigated were methylene blue, thionine, daunomycin and adriamycin. Absorbance-based melting curve analysis revealed a significant increase of the melting temperature of duplex DNA in the presence of oxidised daunomycin. This was not observed in the presence of chemically reduced daunomycin, which confirmed the hypothesis that switching of the redox-state of daunomycin altered its properties from DNA binding to non-binding. Accordingly this altered the thermodynamic stability of duplex DNA. The difference in the stability of duplex DNA, as a direct result of the redox-state of daunomycin, was exploited to drive cyclic electrochemically controlled DNA denaturation and renaturation under isothermal conditions. This proof-of-principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis with in situ UV–vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80 % of the duplex DNA was reversibly hybridised. Five cycles of DNA denaturation and renaturation were achieved and gel electrophoresis as well as NMR showed no degradation of DNA or daunomycin. As no extreme conditions were implicated, no covalent modification of DNA was required and isothermal conditions were kept, this finding has great potential to simplify future developments of miniaturised and portable bioanalytical systems for nucleic acid-based molecular diagnostics