Development of CRISPR/Cas12a-based novel biosensors for non-nucleic acid detection

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Embargoed until 2024-04-27
Copyright: Li, Yi
Abstract
CRISPR/Cas9-based gene editing is no doubt among the most intensively studied topics in bio-related fields in the recent decade, and certain new programmable Cas nucleases have been exploited recently for the development of a variety of biological tools far beyond gene editing, particularly for biosensor development. Although CRISPR/Cas-based biosensing has brought about a revolution in the area of nucleic acid diagnostic with their superior performances, its advantages were challenged when attempting to expand towards a broader range of non-nucleic acid targets. The currently reported methods for non-nucleic acid targets has successfully demonstrated the versality of CRISPR/Cas components in combining with other biosensing elements, however, combining these elements without proper optimization or controllable bioreaction environments could also bring additional variable factors into the system, hence potentially leading to compromised sensitivity or overall increase of system complexity. In this project, two novel CRISPR/Cas12a-based biosensing systems have been developed to realise simple, sensitive and universal non-nucleic acid detections. Both of these systems are established on a standard 96-well plate format, similar to the widely used ELISA diagnostic approach. The first system utilised an aptamer as its recognition molecule for rapid detection of two small proteins with fM-level sensitivity within 1.5 hours. In the second system, antibody was used as a recognition molecule, allowing to draw on a huge pool of commercially available antibodies to support its universality. This system exhibits ultra-high sensitivity down to 1 fg/mL (aM-level) for the detection of two protein targets. With these two successfully developed CRISPR/Cas12a-based non-nucleic acid biosensing systems, the universality and feasibility to deal with two practical bio-detection scenarios was investigated. The antibody-based system with minor modifications was directly used as a ready-to-use sensitivity enhancer onto a commercial IFN-γ ELISA kit, which resulted in a 2-log increase in sensitivity without changing its original protocol. Then, a similar modification strategy was used to re-direct the antibody-based system to detect whole pathogenic microorganism, Cryptosporidium parvum oocyst. Without the need for specialised instruments, the results show a successful detection of this pathogen with single oocyst sensitivity and capable of applying in challenging environmental samples. All these results serve as successful demonstration of the great potential in CRISPR/Cas-based biosensing technology to achieve affordable, translatable, and deployable solutions for various clinical, industrial and research diagnostic needs.
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Publication Year
2022-01-31
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Thesis
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PhD Doctorate
UNSW Faculty