Institute of Genomics & Integrative Biology, Delhi CSIR-National Chemical Laboratory, Pune.
Symposium: “Rare Genetic Disorders” organized by Sudha Bhattacharya, JNU, New Delhi
FELUDA$^SCD$ - FnCas9 editor linked uniform detection assay for sickle cell disease
Sickle cell disease (SCD) is a rare blood disorder that is inherited in an autosomal recessive manner. This is a life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. My presentation will describe a method for using a bacterial CRISPR Cas Ribonucleoprotein complex for detecting single nucleotide variants SCD, without the need for sequencing. The principle of discrimination is derived from the natural property of the enzyme being used for the invention, Francisella novicida Cas9 (FnCas9) which shows very low binding affinity to mismatched substrates. DNA is isolated either from blood, saliva. The DNA is subjected to polymerase chain reaction, amplifying using specific primers and tagging the amplified DNA products with a ligand of choice. The detection mix consists of labelled PCR products, sgRNA-fnCAS9 complex. The detection complex can be visualized using a wide array of technologies like lateral flow, gel based cleavage assay, fluorescence based detection, in both low, medium or plate based high-throughput format. The most important advantage of the present invention as a detection tool over closest prior art is the combination of speed, reliability, robustness and universal applicability for all DNA and RNA variations. Acknowledgement: CSIR-Sickle Cell Disease Mission Mode Project.