The current standard mode of Bd and Bsal detection is qPCR. In our iGEM project, we introduce a novel detection technique by coupling an isothermal amplification reaction with CRISPR detection. Hypothetically, this coupled system will significantly increase the specificity of our detection technique. Subsequently, our engineering team will be integrating this detection technique into a field-ready point-of-care device that will detect the presence of the fungal infection within 30-60 minutes. The ultimate aim of our detection system is to identify the best combination of amplification and CRISPR/Cas enzymes in order to give the most sensitive and specific detection technique. In current literature, there are numerous amplification methods and two particular CRISPR enzymes - CRISPR/Cas12a and CRISPR/Cas13. Our brainstorming process included the necessary research and experimental planning for.
The Cas12a protein is a DNA targeting enzyme that programmatically binds and cleaves DNA. The guide RNA is a complementary sequence to the target DNA. The guide RNA helps the Cas12a enzyme to recognize the target sequence and activates the Cas12a enzyme. The CRISPR/Cas12a system recognizes a T-rich protospacer-adjacent motif (PAM) that catalyzes the maturation of its guide RNA and dsDNA break with staggered 5’ and 3’ ends. The activated Cas12a enzyme unleashes cis (for bound DNA) and trans (for other ssDNA in the solution) cleavage activity. This non-specific trans-cleavage activity can cleave a quenched fluorescence reporter (DNA sequence) in our detection medium to indicate the presence of the targeted nucleic acid sequence. Coupling CRISPR/Cas12a with RPA gives rise to a novel detection technique called DNA endonuclease-targeted CRISPR trans reporter (DETECTR) that can achieve attomolar sensitivity. The DETECTR method specifically uses LbCas12a as its CRISPR enzyme, which comes from Lachnospiraceae bacterium.
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The reporting mechanism of our detection system will rely on the CRISPR enzyme's ability to activate non-specific trans-cleavage activity upon detection of the target gene. We will incorporate a nucleic acid quenched reporter molecule in our detection system such that upon detection, our CRISPR enzyme will indiscriminately cleave the reporter molecule and produce a readout that is detectable through the naked eye. In our research, we narrowed down our reporting mechanism to two different techniques: a quenched fluorescence reporter molecule, and a lateral flow immunoassay with a FAM biotin reporter.
Detection system workflow: from amplification to detection to fluorescence signaling.
Li et al. 2019 Kellner, 2019
