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Enabling CRISPR-Cas mediated nucleic acid detection at Deakin University
thesisposted on 2023-06-06, 23:46 authored by Bailey Siwek
Infectious diseases have been a global health threat in the past, present and future. Access to rapid, sensitive and cost-effective diagnostic tools is one of the essential elements in effective disease management. Polymerase Chain Reaction (PCR) is the current gold standard in molecular diagnostics based on its sensitivity and specificity. However, its predominant use in specialist centralised facilities limits the time to result in developed countries while making the technique merely inaccessible for developing countries. Recently, isothermal amplification methods have been coupled with a sensitive and accurate sequence recognition read-out, re-presenting an exciting opportunity in point-of-care diagnostics. The read-out employs Clustered Regularly Interspaced Short Palindromic Repeats (CRISRP) and CRISPR Associated Protein (Cas) systems including Cas12a, Cas12b, Cas13a and Cas14a. For example, the ‘SHERLOCK’ CRISPR-Cas12b diagnostic platform combines isothermal amplification with fluorescent detection facilitated by a thermostable ortholog of Cas12b found in Alicyclobacillus acidophilus bacteria (AapCas12b), which has previously been applied for detecting zika virus, dengue fever and COVID-19, allowing for detection within one-hour. Unfortunately, access to the promising Cas enzyme remains limited in Australia. To enable the development of Cas12b-mediated detection, a method of recombinant protein expression and purification in Escherichia coli (E. coli) was developed to produce AapCas12b. This research advanced earlier reports by introducing alternate fusion protein tags and using alternate cleavage proteases, increasing the expression yield by 5 times. The purified AapCas12b was combined with isothermal amplification and used to facilitate fluorescent detection comparable to previous literature. In addition to the improvement in protein isolation, this research has decreased the reliance on the commercial availability of Cas enzymes and is expected to enable new research in Australia.