Apart from other CRISPR-based diagnostics methods that detect DNA and RNA, this assay with the help of CRISPR-Cas12a and bacterial allosteric transcription factors (#aTFs) promises to detect the nucleic acids as well as small molecules like Uric acid from human samples. This supersensitive, simple, fast and high-throughput platform is named as #CaTSMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector) assay.
Principle:
The basic principal at play here revolves around two components: the #cellulose-binding
domain (CBD–aTF) and #Cas12a–crRNA complex. The CBD–aTF is bound to microcrystalline #cellulose and immobilized on the sample platform. Now lets say a #dsDNA molecule with a binding motif corresponding to the aTF and a PAM corresponding to the #crRNA is being analyzed. The #aTF will bind to the DNA by its DNA-binding domain first, but after conformational changes the DNA will be dislodged and bind to the Cas12a-crRNA complex through #PAM. This will trigger the nonspecific #ssDNA trans cleavage activity of Cas12a and subsequent reporting of the event through #fluorescence.
Protocol:
FIG: CaT-SMelor assay protocol.
The protocol is simple and quick basically in sync with other Cas-based detection assays.
Merits:
One of the most important advantage is it can be used to detect biochemical parameters too other than DNA/ RNA.
This is cheap with cost per reaction in the range of <$0.3, much cheaper than #qPCR (>$2) or #RPA (>$100).
#TAT is among the fastest with only 15-25 mins needed to generate results.
Limit of Detection (#LOD) is in the nM level.
Demerits:
Development of each assay could be expensive.
Applications:
The applications of the technology at hand will only find its way to medical and food #diagnostics.
More to chew:
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