Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification

Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives

“…Another Cas13a cascade cyclic reaction based on a similar nucleic acid circuit principle also showed a low LOD of 75 aM. 124 After the target RNA recognized by the first Cas13a, it can non-specifically digest fluorescent reporters as well as one designed mediator molecule. The mediator came with double-stranded RNA with a toehold, and it could activate another Cas13a effector after being first digested.…”

Target-triggered CRISPR–Cas13a autocatalysis-driven amplification strategy for one-step detection of circadian clock gene

“…In this context, the TSRP should possess two functions: (1) effectively blocks the RNA intermediate binding to crRNA2, which is essential to minimize the nonspecific background signal; (2) effectively releases the RNA intermediate in response to the BMAL1 target, which is essential to maximize the signal output. In addition, LbuCas13a was reported to recognize ssRNA rather than dsRNA, and its collateral cleavage activity displayed highly base preference with an order of U > G > A = C. 22 Based on the above considerations, two inhibitor RNAs were employed to lock the RNA intermediate. As shown in Fig.…”

“…To improve detection sensitivity, Cas13a has been combined with various nucleic acid-based signal amplification strategies. [21][22][23][24][25] However, these integrated systems may suffer from the timeconsuming procedures, requirement of specialized equipment and careful probe design, as well as pre-amplification of RNA. Autocatalysis, a process vital to biochemistry and mimicking physiological mechanisms, has recently been utilized in nucleic acid-based circuits for detecting diverse biomarkers.…”

CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives

“…On the basis of the same principle, Zeng et al used Cas13a to realize amplification-free detection of miRNA-21. 41 The changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM. Furthermore, the sensitivity can also be improved by designing specific crRNAs at multiplexed sites of targets.…”

“…There is another Cas13a cascade reaction according to the above principles, which was reported by Zeng and coworkers. 41 They harnessed a hairpin RNA mediator that served as substrate for the first cleavage of activated Cas13a. More strikingly, the mediator contained a toehold that could trigger another Cas13a effector after being first digested.…”