CRISPR-based biosensing of non-nucleic acid targets

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has fundamentally transformed life science research in the last twenty-five years. Beyond their initial breakthrough in genetic manipulation, the precision and versatility of CRISPR-based techniques, combined with the advantages of nucleic acid-based nanotechnology, hold great promise in transforming the landscape of molecular diagnostics. Despite the considerable advancements in CRISPR-based diagnostics, notably in the high-sensitivity, programmable, and simple detection of nucleic acids, there exists a potential limitation in their applicability to other biomolecules. To expand the applicability and fully leverage the advantages offered by CRISPR-based diagnostics, ongoing efforts explore the combination of innovative molecular strategies to develop CRISPR sensors capable of detecting a diverse range of analytes beyond nucleic acids. In addition, sensitivity improvement strategies including the optimization of CRISPR enzymes and reporter systems, as well as the incorporation of pre-amplification methods have been widely considered. This PhD thesis delves into CRISPR-based sensing, with a primary focus on two critical aspects: enhancing the sensitivity of pre-amplification-free CRISPR-based assays and expanding detection capabilities beyond nucleic acids, emphasizing the associated design principles. Following an introduction to CRISPR/Cas systems and their applications in biosensing, first, I introduce hairpin DNA sequences as superior FRET-based reporters of CRISPR/Cas12a-based sensors (Chapter 2). Hairpin DNA probes significantly enhance FRET-based signal transduction compared to the widely used linear single-stranded DNA reporters, leading to improved sensitivity and specificity for nucleic acid detection. In Chapter 3, I detail the design of a new class of regulators of Cas12a, namely PAM-engineered Toehold Switch DNA. They present a re-engineered locked protospacer adjacent motif (PAM) in the loop that enables the fine control of Cas12a activities in response to the specific molecular target through structure switching and PAM complementation. PAM-engineered Toehold Switch DNA reconfiguration can be rationally controlled by a proximity-based reaction network to achieve single-step detection of diverse targets (e.g., IgG antibodies, small molecules, and microRNAs) with high sensitivity and specificity even within complex matrices. Chapter 4 presents an innovative strategy for monitoring protease activity (i.e., matrix metalloproteinase 2 (MMP2)), integrating an activity-based method and Cas12a-assisted signal amplification. The design involves a chimeric peptidePNA conjugate serving as a molecular translator, converting a protein input into a nucleic acid activator for Cas12a. This enables highly sensitive detection of diverse proteins and enzymes. Furthermore, in Chapter 5, the minimalist design of robust wireframe DNA nanotubes is described, a project undertaken during my visiting period in the laboratories of Prof. Sleiman at McGill University where I challenged my expertise with a new research topic. Specifically, I report here on the implementation of a DNA-RNA hybrid nanotube design that allows for precise enzyme-mediated control over nanotube assembly and disassembly rendering them promising candidates for multivalent biosensing scaffolds and drug delivery vehicles for biomedical applications. In conclusion, the result of this PhD thesis not only contributes to the expanding body of knowledge on CRISPR-based technologies but also provides a comprehensive exploration of their potential in revolutionizing biosensing for non-nucleic acid targets.