The advancements of #CRISPR-based diagnostics in the past 2 years have been phenomenal but it could not do away with the #PCR step, until #AIOD (All In One Dual) CRISPR Cas12a assay came into the scene. As with #SHERLOCK, #DETECTR and #HOLMES assays, there are multiple manual operations along with amplification steps (#PCR, #RT-PCR, #qPCR, #LAMP, #RPA etc.) which increases the chances of complications, contaminations and errors in the assay.
Principle
The fundamental difference here from other CRISPR Dx is the use of two different Cas12a-crRNA complexes formed by different crRNA sequences corresponding to the recognition sites of frorward and reverse primers in the target sequence. The Cas12a-crRNA complexes are supported by Recombinase, RPA-specific primers, ssDNA-FQ reporters (5'-TTATT-3', labelled by 5' 6-FAM (Fluorescein) fluorophore and 3' Iowa Black® quencher), single-stranded DNA binding protein (#SSB), strand-displacement DNA polymerase and the target DNA sequence (in case of target RNA sequence, we have to have AMV RT enzyme in the cocktail). The RPA amplification at 37C displaces the DNA strand and exposes the Cas12a-crRNA binding sites. On successful recognition and binding of the crRNAs, the collateral cleavage activity of the #Cas12a is triggered. This causes indiscriminate cleavage of ssDNA-FQ reporters and consequent rise in fluorescence. The RPA-amplified products also serve as substrates to Cas12a-crRNA binding, thereby amplifying the fluorescence result.
FIGURE: Working principle of AIOD CRISPR Cas12a assay.
Protocol
AIOD CRISPR Cas12a assay has the simplest protocol among all the CRISPR Dx platforms. This one-tube reaction is completed in ~40 mins and efficiently produces results in a visual readout.
The reaction components are prepared separately as 3 different components:
TABLE: Required reagents and their strength are mentioned here for ready reference in developing similar assays, although standardization is highly recommended.
The mix is incubated at 37C for 40 mins and the color change of the sample from orange/yellow to green signs positive, although UV based readout is also possible.
FIGURE: Different possible result readout methods for AIOD CRISPR assay
Importance
1. The AIOD CRISPR Cas12a system is a true single tube reaction system which avoids pre-amplification of target nucleic acid, physical separation of Cas enzyme or any other special requirements.
2. This is a true #isothermal detection system with One Pot, One Step, One Temperature working principle.
3. This system has the fastest #TAT (~40mins) among all other CRISPR Dx systems.
4. This is a highly sensitive assay with reports of detecting #SARSCoV2 and #HIV1 to the tune of 4.6 and 1.2 copies of viral #RNA.
5. This is a very specific assay as well with comparable signal-to-noise ratio among its contemporaries.
6. With addition of #AMV RT enzyme it can easily detect pathogens from RNA, circumventing the #cDNA preparation step at no additional time.
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