Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity

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

“…In the absence of the target, the Apt-acDNA hybridizes to the cDNA immobilized on the microplate, which then activates Cas12a upon acDNA hybridizing with crRNA. The activated Cas12a cleaves a fluorescent ssDNA reporter labeled with both a fluorophore and a quencher, producing high fluorescence signals. ,, When a small molecule target exists, the Apt-acDNA probe binds with the target and forms a stable conformation, inhibiting hybridization between the Apt-acDNA probe and cDNA on the microplate, which leads to a decrease in the amount of Apt-acDNA probes captured on the microplate. Thus, less Cas12a is activated and the fluorescence signal is reduced.…”

“…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.…”

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

“…In addition, preconcentration of targets may be achieved in the immunosorbent assays by affinity capture with the immobilized antibody to further lower the detection limits. The sensitivity of this assay can be further improved by employing optimal DNA reporters. , …”

“…The sensitivity of this assay can be further improved by employing optimal DNA reporters. 47,48 ■ CONCLUSIONS In summary, we have developed a CRISPR/Cas12a powered competitive immunosorbent assay for the sensitive detection of small molecules. This method integrates target recognition by antibody with high affinity, easy modification of DNA, effective signal amplification by trans-cleavage activity of CRISPR/ Cas12a, and the high-throughput format of the microplate assay.…”

Exploring the Effect of Steric Hindrance on Trans-cleavage Activity of CRISPR-cas12a for Ultrasensitive SERS Detection of P53 DNA

“…20 Researchers have also focused on optimizing reaction conditions such as adjusting the Cas12a/ crRNA ratio and changing the buffer to improve its cleavage activity. 19 Recently, its reporter gene substrate has also been explored, its cleavage efficiency accelerates with increasing substrate density, 21 and the hairpin reporter has a higher affinity for cas12a relative to the single-stranded reporter gene. 22 Nowadays, some literature studies point out that spatial site resistance affects its cleavage activity, but few studies have explored the effect of spatial site resistance on its cleavage activity.…”