Next-Gen CRISPR Diagnostics

Researchers at the RNA Biology Lab have developed diagnostic strategies via the CRISPR-Cas system. The high specificity and sensitivity of the system make it a valuable tool for diagnostics, particularly for identifying pathogenic or mutational signatures. FnCas9 (Cas9 isolated from Francisella novicida) due to its extremely high base mismatch sensitivity, has been adapted in the lab for the development of the FnCas9 Editor Linked Uniform Detection Assay (FELUDA). it acts as a point-of-care diagnostic kit for the detection of SARS-CoV2 variants during the COVID-19 pandemic. The kit can also successfully detect HBB variants in SCD patients and

The endonuclease FnCas9 is guided by a gRNA that can recognize the target genome. Complementarity leads to the binding of the ribonucleoprotein complex to the region of interest. The output can be read through multiple signal detection platforms and thus can be used for rapid diagnosis of both genetic as well as pathogenic signatures. For instrument-free visual detection, FELUDA has been adapted to be performed on lateral flow strips that capture this complex and result in a clear test band, called Rapid variant Assay (RAY). For this, FAM-labeled RNP complex (biotinylated amplicon DNA-FAM labeled sgRNA bound FnCas9) and biotinylated amplification products have been used. The complex could be visualized as it gets captured by streptavidin present on the strip test line. This method has been applied to developing a rapid, cost-effective, and instrument-free paper strip test for COVID-19. Currently, this technology has finished licensing, and approvals and is available commercially. Together with the lab of Dr. Souvik Maiti, we plan to modify the diagnostic strategy so that it can distinguish carriers from homozygotes for various genetic diseases. A web-based tool, JATAYU (Junction for Analysis and Target Design for Your FELUDA assay) has been created for the design of sgRNA and primers using FELUDA making it easily adaptable for the detection of many mutational variants.

RNA BIOLOGY LAB

Next-Gen CRISPR Diagnostics