Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity

Abstract

Existing CRISPR/Cas-based biosensors usually improve sensitivity by target amplification, which is time-consuming and susceptible to the impurities in complex biofluid. Herein, it is the first time to develop a cascade...

CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

“…Recently, Sha et al developed a sensitive and specific CRISPR-Cas-based biosensor termed casCRISPR for rapid and accurate miRNA detection, without a target amplification process (e.g., polymerase chain reaction (PCR) or recombinase polymerase amplification (RPA)), even in cell extracts and serum samples [ 43 ].…”

“…In this regard, the phosphodiester bond next to the rU of the “locked-trigger” for Cas14a/sgRNA is cleaved ( Figure 2 ). The latter makes the “locked-trigger” transform from a stable hairpin structure into a duplex structure with a 5′-toehold, which can initiate the interaction with Cas14a/sgRNA through strand displacement [ 43 ]. Once the “locked-trigger” is cleaved, the trans -cleavage activity of Cas14a is elicited, resulting in efficient separation of the fluorophore (6-FAM) and the quencher (BHQ1) on the ssDNA reporter sequence, thus providing the fluorescence signals [ 43 ] ( Figure 2 ).…”

“…Employing the trans -cleavage activity of Cas13a and Cas14a, the casCRISPR system can achieve miR-17 detection with a limit of detection on the order of 1.33 fM (~1000 times lower than direct Cas13a-based miRNA detection), displaying single-base specificity. Due to the Cas13a/crRNA high-fidelity recognition ability, casCRISPR could provide higher specificity for miRNA detection without the target amplification required by the qRT-PCR method, thus becoming a promising tool for miRNA diagnostics [ 43 ].…”

“…Since Cas12a also can be behave as ssDNA-activated DNase [ 21 ], the authors constructed another version of casCRISPR-coupled Cas13a with Cas12a (casCRISPR v2) comparing its performance to casCRISPR v1 (Cas13a–Cas14a) [ 43 ]. The fluorescence intensity curves obtained by these platforms demonstrated that casCRISPR v1 could detect miR-17 as low as ~6.25 fM, while casCRISPR v2 could detect ~100 fM miR-17.…”

“…The fluorescence intensity curves obtained by these platforms demonstrated that casCRISPR v1 could detect miR-17 as low as ~6.25 fM, while casCRISPR v2 could detect ~100 fM miR-17. However, since Cas14a possesses higher cleavage efficiency than Cas12a when ssDNA is used as the activator, this could explain why Cas13a–Cas14a (casCRISPR v1) can achieve a lower detection limit and a higher signal to background ratio than Cas13a–Cas12a [ 43 ].…”