Oh yes! if we are to believe Dr. Souvik Maiti and Dr. Debojyoti Chakraborty of #CSIRIGIB, they have come up with a less than Rs. 500 assay to detect #SNP, named after the #SatyajitRay sleuth #Feluda. The Fn (Francisella novicida) Cas9 Editor Linked Uniform Detection Assay (#FELUDA) which uses the FnCas9 protein and a lateral flow paper-based strip is now awaiting approvals to be used for detecting #COVID19. Although this concept of #CRISPR diagnostics (#CRISPRDx) is only about 2 years old of employing different Cas enzymes to specifically detect nucleotide sequences, it surely holds promise.
FnCas9 enzyme
The CRISPR associated protein 9 (Cas9) is a part of bacterial immunological defense against many DNA/RNA viruses. It is in effect an endonuclease in a four-component defense system including two small RNA molecules named CRISPR RNA (#crRNA) and trans-activating CRISPR RNA (#tracrRNA) and of course the target nucleic acid. Outside of the bacterial system, #crRNA and #tracrRNA can be fused into a "single-guide RNA" (#sgRNA) that, when combined with #Cas9, could find and cut the DNA target specified by the guide RNA. #Francisella_novicida Cas9 (FnCas9) which is used in FELUDA, shows a very high specificity of binding to its intended targets and negligible binding to off-target loci as compared to SpCas9 or other genetically engineered counterparts. The specificity is evident from its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex, making it an ideal choice for this assay.
FIG: FnCas9 working principle #FnCas9
Principle
The scientists found out that if there is a mismatch between the sgRNA and analyte DNA sequence at positions 2nd and 6th upstream of the NGG PAM, the analyte DNA is not cleaved, whereas if there is only a single mismatch either at 2nd or 6th position, FnCas9 will cut the DNA. For designing the sgRNA they have created a webtool, #JATAYU (Junction for Analysis and TArget design for YoUr FELUDA assay).
FIG: Specific DNA cutting by FnCas9 under the presence of two different sgRNAs.
Detection of cleaved DNA
Although routine gel electrophoresis could deliver the result, a fluorescence-based approach was developed to hasten the process. The proposed assay uses #FAM-labelled RNP complex (with a deactivated FnCas9 that cannot cleave) and #biotin labelled amplicons/ substrates (generated by PCR with biotin-labelled primers) on commercially available #LateralFlowStrips. Here the RNP-bound substrate molecules in a solution can lead to aggregation of the complex on a distinct test line of the strip having #streptavidin coating. On the other hand, anti-FAM antibody linked gold nanoparticles accumulate on a control line on the strip.
FAM labelling of sgRNA had to be followed by validating successful activity of chimeric dFnCas9 (deactivated FnCas9) sgRNAs by altering the length of overlap between crRNA:tracrRNA. It was found that 19nt overlap can efficiently cleave the target, and since a single FAM labelled tracrRNA is compatible with multiple crRNAs, this design also reduced the time and cost of a FELUDA assay.
FIG: LFD-based detection strategy
Significance of FELUDA
For genotyping individuals with high confidence, including carriers of a single nucleotide variant (SNV), Cas12a (#DETECTR) requires individual sgRNA design and optimization for every variant and Cas13 (#SHERLOCK) requires conversion to an RNA template prior to detection, both of which increases the complexity and thus the time taken for diagnosis. But FELUDA employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision to single nucleotide.
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