Periodic Reporting for period 1 - SCOPE (CRISPR Point-of-Care Diagnostics)
Reporting period: 2022-09-01 to 2024-02-29
CRISPR-Cas can be used in a diagnostic setting to detect specific DNA or RNA sequences. The current SCOPE platform consists of two steps, which impedes its usability due to complexity and a higher risk of contamination (Steens et al. 2021)).
The extra step is required because conceptually the reaction consists of a amplification and detection step. To connect these two steps, a thermostable RNA polymerase is required, which is not commercially available and thus needs to be added in a second step. In this project, a newly discovered RNA polymerase is tested and characterized to integrate this protein into the SCOPE assay, creating a one-pot reaction resulting in SCOPEv2. This diagnostic assay can be used to detect pathogens at an early stage of infection in a quick and simple manner.
The extra step is required because conceptually the reaction consists of a amplification and detection step. To connect these two steps, a thermostable RNA polymerase is required, which is not commercially available and thus needs to be added in a second step. In this project, a newly discovered RNA polymerase is tested and characterized to integrate this protein into the SCOPE assay, creating a one-pot reaction resulting in SCOPEv2. This diagnostic assay can be used to detect pathogens at an early stage of infection in a quick and simple manner.
The phage-derived RNA polymerase PhiFa-44 was identified and characterized in vitro with the use of recombinant protein expression.
PhiFa-44 requires dsDNA as a template and divalent metal ions, particularly Mn²⁺ and Co²⁺. The optimal temperature for activity is 35-40°C, surprisingly below the optimal growth temperature of its host. However, the activity at the required temperature for the SCOPE platform (65°C) is still sufficient for the intended purpose.
The optimal pH for PhiFa-44 activity was found to be 6.7-8.6. PhiFa-44 can be used in the current SCOPE platform without affecting the assay, and it still maintains sufficient RNA polymerase activity to enable a one-pot reaction (SCOPEv2).
This newly developed one-pot reaction composition was verified on P. aeruginosa samples. P. aeruginosa is a pathogen that can cause a variety of infections, including respiratory infections, urinary tract infections, and sepsis, particularly in individuals with weakened immune systems (such as COPD patients).
The SCOPEv2 can detect its target within 30 minutes and can detect as little as 10 synthetic copies/µL. Detection of live organisms resulted in a detection limit of 2.5E0 colony-forming units (CFU) per µL, and 2.5E-3 CFU/µL if the sample is pre-boiled.
These results have been made into a draft manuscript and are already published as a chapter in a PhD thesis.
PhiFa-44 requires dsDNA as a template and divalent metal ions, particularly Mn²⁺ and Co²⁺. The optimal temperature for activity is 35-40°C, surprisingly below the optimal growth temperature of its host. However, the activity at the required temperature for the SCOPE platform (65°C) is still sufficient for the intended purpose.
The optimal pH for PhiFa-44 activity was found to be 6.7-8.6. PhiFa-44 can be used in the current SCOPE platform without affecting the assay, and it still maintains sufficient RNA polymerase activity to enable a one-pot reaction (SCOPEv2).
This newly developed one-pot reaction composition was verified on P. aeruginosa samples. P. aeruginosa is a pathogen that can cause a variety of infections, including respiratory infections, urinary tract infections, and sepsis, particularly in individuals with weakened immune systems (such as COPD patients).
The SCOPEv2 can detect its target within 30 minutes and can detect as little as 10 synthetic copies/µL. Detection of live organisms resulted in a detection limit of 2.5E0 colony-forming units (CFU) per µL, and 2.5E-3 CFU/µL if the sample is pre-boiled.
These results have been made into a draft manuscript and are already published as a chapter in a PhD thesis.
SCOPEv2 is now a simple and quick detection tool that can identify the smallest amounts of genetic material (DNA or RNA) with minimal handling steps. This allows for on-site use of the assay to detect pathogens, potentially at an early stage of infection. Each new target will require tweaking of the established assay to ensure specificity for that target, which is a relatively quick process. New assays can be developed in as little as four weeks. The application of this diagnostic platform is broad, encompassing healthcare (pathogen detection) and agriculture (pathogen detection and strain identification). The next steps will be to demonstrate this product in real-life cases to build up a portfolio and enable commercialization. Together, progress made in this project has also lead to the successful application of an EIC Transition project (ongoing since March 2024).