Rapid, accurate, nucleobase detection using FnCas9

Abstract

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.

RAY: CRISPR diagnostic for rapid and accurate detection of SARS-CoV2 variants on a paper strip

“…To develop RAY for SARS-CoV2 variant identification, we first analyzed the mutations arising in the three variant lineages reported from the UK, South Africa, and Brazil and looked for the accessibility to FnCas9 enzyme based on the presence of an NGG PAM site in the vicinity 37 . In an earlier study, we had successfully established that FnCas9 is unable to bind or cleave targets having two mismatches -in the 2nd and 6th position (PAM proximal) of the sgRNA-with respect to the target (Figure 1A) 25,38 . Besides these, we had also systematically identified other regions in the sgRNA where mismatches lead to weak or no substrate binding.…”

“…To generate a visually distinctive signal between WT and mutant variants, we modified our previous FELUDA protocol to identify SNV accurately in the sample 25 . Firstly, we performed a single-step reverse transcription PCR to generate a biotin-labeled amplified product that can be detected by a single sgRNA (S-gene) if the sample is WT and by both sgRNAs (S-gene and N501Y) if the sample contains the N501Y variant.…”

An Update on Molecular Diagnostics for COVID-19

“…A highly accurate, single nucleotide variant detection system developed by IGIB India employs Francisella novicida (Fn-Cas9) based enzymatic readout for nucleotide detection and nucleobase identification. They have named this approach as FnCas9 Editor Linked Uniform Detection Assay (FELUDA) (Azhar et al, 2020).…”

HTA of FELUDA diagnostic test for COVID-19

“…11 This test is based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and was successfully innovated to be used as a diagnostic tool for SARS-CoV-2. 12 While the method of swab collection in this test remains the same as that of RT-PCR and RAT, the results of this test can be read with the naked eye like a pregnancy test within 45 minutes. This innovative diagnostic test has been reported to be 96% sensitive and 98% specific.…”