Development of CRISPR-based diagnostics for point-of-need detection of arboviruses and high-consequence pathogens

Funding period: 2023–2025
Lead: Alexander Bello
Total GRDI funding: $1,020,900

To provide patients with appropriate treatment or to control outbreaks caused by highly infectious agents, rapid diagnostics must be performed to accurately identify the pathogen. Rapid antigen detection provided by lateral flow (LF) is fast, cheap, user-friendly, and stable at room temperature but comes at a cost of decreased sensitivity and assay development is a long process before distribution. Alternatively, molecular diagnostics, such as the gold-standard polymerase chain reaction (PCR), are highly sensitive and specific and rapidly adaptable to emerging pathogens, but requires expensive equipment, skilled personnel, cold storage, and has longer turn-around-times. An ideal diagnostic tool would have the best qualities of both LF and PCR with none of the drawbacks; CRISPR-based diagnostics encompasses the strengths of both LF and PCR in that CRISPR-based assays are inexpensive, fast, sensitive, specific, user-friendly and can be rapidly adapted to new emerging pathogens.

Previously, we were awarded a proof-of-concept GRDI Round 7 grant to develop CRISPR-based diagnostics for detection of high-consequence pathogens, where we showed the technology could be used to detect Ebola virus in a single-plex reaction on a fluorescent reader. However, within the first year of funding the COVID-19 pandemic caused us to shift our resources and priorities to the pandemic response and by using CRISPR-based technology, we were able to adapt this to detect SARS-CoV-2. Since then, newer CRISPR-Cas enzymes have been identified that can be used simultaneously to detect multiple targets in a multiplex reaction, adding to the CRISPR-based diagnostics toolbox. For the current project proposal, our goal is to develop a deployable paper-based CRISPR-based diagnostic assay using newer or engineered enzymes to rapidly detect arboviruses or high-consequence pathogens, be it at the bedside, in the field, or on the diagnostic bench. In resource limited settings such as Canadian northern, remote, and isolated (NRI) communities, providing access to rapid, low-cost CRISPR-based diagnostics will vastly improve patient health in these remote areas. In acute care settings, returning travellers who present with fever and rash from areas of the world endemic for arboviral pathogens such as dengue or chikungunya virus, but also high-consequence pathogens such as Ebola or Lassa virus must be provided with rapid diagnosis for the patient to be provided appropriate care or isolation requirements.

We will also develop bioinformatics tools for design of primers for pre-amplification of template, as well as design of CRISPR guide RNA. For the current full project proposal, implementation of CRISPR diagnostics at the National Microbiology Laboratory (NML) and at the point-of-need will lead to faster and cheaper diagnostic tools, translating to cost savings for taxpayers and healthier Canadians.