A versatile biosensing platform coupling CRISPR–Cas12a and aptamers for detection of diverse analytes

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection

“…, spike (S), nucleocapsid (N), membrane (M), and envelope (E), of which S protein and N protein have been used in antigen detection [ 3 , 26 , 27 ]. In this study, to couple antigen recognition with a CRISPR-based detection system, we used a specific aptamer (A48, with an equilibrium dissociation constant ( K D ) of 0.49 nM) [ 27 ] of N protein as the recognition element to develop a SARS-CoV-2 antigen biosensor with our recently established CRISPR/Cas12a-based biosensing platform [ 15 ]. Our prototype was denoted the antigen biosensing platform CaT-Smelor-Covid.v1 (CRISPR/Cas12a and aptamer-mediated detector of diverse analytes for COVID-19, version 1).…”

“…In the CRSIR/Cas12a and FQ-based signal output and display modules, the slopes of the fluorescence signal are proportional to the concentration of the released triggering dsDNA [ 15 ]. To increase or amplify the signal generated by the Cas12a triggering dsDNA, we designed a cluster of multiple triggering dsDNAs in tandem.…”

“…In parallel to the nucleic acid detection systems DETECTR, SHEROLOCK, and STOPCovid, the combination of CRISPR/Cas12a and aptamers represents a new detection paradigm for antigen detection [ [34] , [35] , [36] , [37] ]. Coupling with aptamers dramatically broadens the detection scope of CRISPR/Cas12a [ 15 , 22 ]. However, how to stably amplify the signals sensed by aptamers and then translate the amplified signal into Cas12a cleavage activity has been an unresolved problem.…”

“…CRISPR/Cas-based nucleic acid detection systems, such as SARS-CoV-2 DETECTR [ 11 ], SHEROLOCK [ 12 ], STOPCovid [ 13 ], and AIOD-CRISPR [ 14 ], have been developed and applied in rapid diagnostic tests for SARS-CoV-2. The advantages of CRISPR/Cas-based detection systems have been well demonstrated, and these methods have shown great potential during the COVID-19 pandemic, owing to their superior speed, portability, low cost, and comparable sensitivity to that of the traditional gold standard of RT-qPCR assays (one copy/μL of viral RNA) [ [15] , [16] , [17] , [18] ]. However, the CRISPR/Cas-based detection paradigm has not been expanded to SARS-CoV-2 antigen detection [ 19 ].…”

“…Previously, CRISPR/Cas12a and aptamer-mediated systems for detection of diverse analytes ( e.g. , proteins or small molecules) have been developed [ 15 , 22 ]. These platforms directly translate the signal of aptamer responsive target molecules into a CRISPR-mediated nucleic acid detection-based output signal.…”