Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor

Detection of SARS-CoV-2 Based on Nucleic Acid Amplification Tests (NAATs) and Its Integration into Nanomedicine and Microfluidic Devices as Point-of-Care Testing (POCT)

“…Sample preparation must be minimal to avoid RNA extraction, which is a time-consuming step and affects sensitivity, depending on the extraction method [ 122 ]. Many of the approaches we have reviewed have taken this into account and can be performed rapidly, thanks to isothermal amplification kinetics and the tolerance of inhibitors, or the specificity of functionalized nanoparticles, such as glass slides or aptamers [ 53 , 66 , 104 , 105 , 108 , 109 , 112 ].…”

“…CRISPR cleavage is detected via the binding aptamer S1, and measured using electrochemical impedance spectroscopy and differential pulse voltammetry, with an ultra-high sensitivity of 1.5 pg/mL. It has been tested in several variants of interest as a promising POCT [ 112 , 113 ].…”

CRISPR/Cas12a-Amplified Aptamer Switch Microplate Assay for Small Molecules

“…Target binding directly inhibits acDNA-crRNA hybridization and Cas enzyme activation. 31,32 Because of the dearth of flexible signal transduction strategies, the current methods require rational engineering of acDNA and corresponding crRNA according to the target small molecule, limiting the application of the CRISPR/Cas system for the detection of a wide range of analytes.…”

“…In the past few years, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), identified as an adaptive immune mechanism in bacteria and archaea, have sparked a research upsurge in the field of biosensing technology. , Cas12a is a CRISPR RNA (crRNA)-guided enzyme, which can cleave target double-stranded DNA (dsDNA) or target single-stranded DNA (ssDNA) within a RuvC catalytic pocket upon its target DNA complementary to the preordered seed region in crRNA (termed cis-cleavage activity). − Particularly, the target DNA-activated Cas12a can indiscriminately cleave surrounding nonspecific ssDNA with high enzymatic efficiency (termed trans-cleavage activity). ,, This unique cleavage property of Cas12a has been widely deployed to construct a series of amplified detection platforms for the analysis of different targets. − ,− For small molecule detection, aptamers are often used as affinity ligands, and a majority of methods utilize a structure-switchable aptamer to regulate the activity of Cas12a responsive to target information. − , In most cases, the single-stranded active DNA (acDNA) of Cas12a is locked by the aptamer, and only the presence of the target triggers the release of acDNA and activation of Cas12a. − Another typical signal-off pattern relies on a rationally designed ssDNA probe, which functions as both a Cas12a activator and a target-binding ligand. Target binding directly inhibits acDNA-crRNA hybridization and Cas enzyme activation. , Because of the dearth of flexible signal transduction strategies, the current methods require rational engineering of acDNA and corresponding crRNA according to the target small molecule, limiting the application of the CRISPR/Cas system for the detection of a wide range of analytes.…”