FnCas9 Editor Linked Uniform Detection Assay or FELUDA, is a CRISPR-Cas based diagnostic test that was developed in collaboration with Dr. Debojyoti Chakraborty for point-of-care detection of Sickle Cell Anemia in low cost settings. It is a rapid, robust, highly specific and sensitive and inexpensive assay that can be adapted with ease for the detection of other diseases ( genetic and infectious) as demonstrated by the short timeline that it took us to adapt FELUDA for the detection of SARS-CoV2 when the COVID-19 pandemic gripped the world.
FELUDA uses a combination of CRISPR-Cas based identification and binding of the sequence of interest (using sgRNA) and lateral flow assay chemistry for visual detection. It uses inactivated FnCas9 to bind to amplified biotin tagged DNA (genomic DNA isolated from saliva of a patient or cDNA produced from isolated RNA for nasopharyngeal swab of suspected COVID-19 patient). The RNP (FAM labeled sgRNA-FnCas9 complex) bound to biotinylated target is captured by using a lateral flow strip that has avidin. The visual detection occurs by the binding of gold nanoparticles conjugated with Anti-FAM antibody to the RNP complex that has FAM labeled sgRNA. Appearance of red colored test band on the strip along with a control band indicates the presence of target sequence in the sample and the assay result is considered to be positive. While a single control band develops in case of a negative result.
The high specificity and mismatch sensitivity of FnCas9 makes the detection extremely robust.
The SARS-CoV2 mutants that predominated in the second wave of the pandemic pushed us to develop a CRISPR-Cas based assay (RAY -- Rapid variant detection AssaY) which relies on a similar principle as FELUDA.