Feluda – A new rapid test method for the identification of SARS-CoV-2
CRISPR-test gets approval in India
Just recently, the TATA Group received approval for the commercial launch of “Feluda” (FNCas9 Editor Limited Uniform Detection Assay) by the Drugs Controller Gerneral of India.
What is Feluda?
Feluda is the first CRISPR/Cas9 rapid test available for COVID-19 detection. With this new qPCR independent point-of-care diagnostic approach, the genomic sequence of SARS-CoV-2 can be detected by Milenias lateral flow assay HybriDetect.
The test was developed at the Council of Scientific and Industrial Research’s constituent lab, the Institute of Genomics and Integrative Biology (CSIR-IGIB). The test addresses the urgent need for accurate SARS-CoV-2 mass testing and has many advantages compared to the gold standard, qPCR. Like SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) which is a COVID test using Cas12 for detection of SARS-CoV-2, developed by scientists from the Broad Institute in the US, the name Feluda refers to an Indian fictive detective.
Advantages of Feluda compared to qPCR:
With 96% sensitivity and 98% specificity, Feluda’s analytical performance is comparable to the qPCR results.
How Feluda works
The test is based on a Cas9 enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without requirement of trans-cleavage activity of reporter molecules like with Cas12 or Cas13 methods.
The scientists from CSIR-IGIB used Cas9 from Francisella novicida (FNCas9), which shows very high mismatch sensitivity and can distinguish between nucleotide sequences differing by only one mismatch. The principle of the test was originally designed for the identification of sickle cell anemia, a disease caused by a point mutation, and adapted to COVID-19 testing due to the urgent need for mass testing.
The FnCas9 used in this new method does not produce collateral activity on substrates due to the use of a catalytically inactive FnCas9-gRNA-complex. Therefore, it is an affinity based method and no trans-cleavage signal output is generated.
Fig. 1: Feluda test principle on HybriDetect. FnCas9 with FAM labeled gRNA binds to biotinylated sample if result is positive.
Feluda and Milenia HybriDetect
The authors designed a gRNA, labeled with FAM, which is important for the visualization of the test line on Milenia HybriDetect (Figure 2).
After RNA extraction, the first step of the Feluda method is an optimized single step Reverse Transctription-PCR, or alternatively a RPA protocol, where the sample gets amplificated and biotinylated.
In the next step, the FnCas9 gRNA (labeled with FAM) is incubated with the biotinylated substate (virus sequence if sample is positive). Due to the streptavidin, immobilized on the HybriDetect dipstick, the RNP Complex (CRISPR ribonucleoprotein) binds to the test line (Figure 2).
The RNP-complex bound to the labeled substrate is visualized with anti-Fam antibodies conjugated to gold nanoparticles and a positive test line is formed if substrate is bound to the RNP-complex.
Fig.2: Milenia HybriDetect and FnCas9. The test is positive if the biotinylated DNA is present and binds to streptavidin on the T-Line. FnCas9 binds to the biotinylated DNA and is captured by FAM antibodies coated with gold nanoparticles for visualization.
Additional Information on Feluda
Compared to other CRISPR methods for COVID Detection, including SHERLOCK and DETECTR, Feluda does not need a reporter and is therefore less complex.
The LOD of the Feluda method is 10 copies of purified viral sequence as the authors mention in their latest publication (https://www.medrxiv.org/content/10.1101/2020.09.13.20193581v1.full.pdf)
To assist the detection of COVID-19 the scientists developed a smartphone app named TOPSE (True Outcome Predicted via Strip Evaluation) which gives a predictive score based on background correction. Due to the stoichiometric binding affinity of FnCas9 RNP to the target, a semi-quantitative readout of Feluda is possible with TOPSE.
Feluda for at home testing
The Scientists from CSIR-IGIB are currently working on a Feluda version for at home testing. Therefore they are trying to do a machine free, RPA based amplification of the virus genome. The aim is an end-to end instrumentation free testing protocol.