UC Berkeley

Molecular diagnostics are an important set of techniques used for the identification and control of infectious diseases, as well as treatment evaluation, and epidemiological surveillance. However, wide adoption of some molecular diagnostic techniques outside of well-equipped laboratory settings has been challenging due to laborious protocols, high cost of reagents, use of sophisticated equipment, and requirement for trained personnel.One promising technique for RNA detection that has the potential to overcome these challenges is based on CRISPR-Cas13a, which is a programmable RNA endonuclease. This dissertation describes my work harnessing Cas13a to sensitively and specifically detect RNA as well as a variety of different targets. Together with colleagues, I (i) established a new method to encapsulate Cas13a reactions into droplets and use variable guide-dependent nuclease activity for multiplexing; (ii) developed a new spatiotemporal light control of Cas13a trans-cleavage activity that can be used to identify viral co-infections; (iii) adapted the Cas13a assay for non-nucleic acid targets and finally, (iv) developed an amplification-free Cas13a assay for detection of lymphatic filariasis, a neglected tropical diseases.I begin by describing encapsulation of Cas13a-based assays into droplets, which enables high sensitivity, quantification of target concentrations, and rapid reaction time. We introduce “kinetic barcoding,” which is based on distinct nuclease activity for different guide and target RNA and can be tuned by introducing an interfering segment to guide RNAs (igRNAs). We exploited this feature to demonstrate multiplexed detection of SARS-CoV-2 variants and influenza viruses.The development of igRNAs, which provide an independent way to regulate nuclease activity, allowed us trigger the start of Cas13a assays using light, a method we called LUCas (Light-Uncaged Cas13a). Moreover, by measuring the kinetic rate of Cas13a before and after simulation, we could calculate a suppression factor for different igRNAs, which are on the order of 100. Using measured kinetic parameters, we predict and experimentally validate the limit-of-detection of the LUCas system and demonstrated a new multiplexing strategy assay called “temporal barcoding,” enabling quantitative detection of viral co-infections in a single bulk reaction.The development of igRNAs also allowed us to develop assays to detect non-nucleic acid targets. We replaced the interfering strand of the igRNA with a DNA aptamer that can bind to specific analytes while maintaining inhibition of the active site of Cas13a. Upon the removal of the DNA aptamer through the addition of the analyte or complementary DNA strand, the trans-cleavage rate increases significantly. We used this approach to detect heavy metals and pesticides in environmental samples.The simplicity of the Cas13a assay raises the possibility of using it for point-of-care applications. As a demonstration, I developed an amplification-free assay to detect active infections of Lymphatic filariasis. I designed and validated guides against RNA extracted from Brugia malayi microfilariae and showed that the fluorescence signal generated by a CRISPR-Cas13a assay can be detected on the NTDScope, a portable multi-contrast microscope. Overall, my work on molecular diagnostics with CRISPR-Cas13a has helped to establish it as a viable alternative to conventional PCR-based assays and opens the possibility of using Cas13a to detect both nucleic acid and non-nucleic acid targets at the point-of-care.