Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs

Abstract

Multiplexed detection of extracellular vesicle (EV)-derived microRNAs (miRNAs) plays a critical role in facilitating disease diagnosis and prognosis evaluation. Herein, we developed a highly specific nucleic acid detection platform for simultaneous quantification of several EV-derived miRNAs in constant temperature by integrating the advantages of a clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases (CRISPR/Cas) system and rolling circular amplification (RCA) techniques. Particularly, the proposed approach demonstrated single-base resolution attributed to the dual-specific recognition from both padlock probe-mediated ligation and protospacer adjacent motif (PAM)-triggered cleavage. The high consistency between the proposed approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived miRNAs’ abundance from both cultured cancer cells and clinical lung cancer patients validated its robustness, revealing its potentials in the screening, diagnosis, and prognosis of various diseases. In summary, RACE is a powerful tool for multiplexed, specific detection of nucleic acids in point-of-care diagnostics and field-deployable analysis.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…B) Schematic of RCA-based isothermal reaction, resulting in ssDNA sequences with a three-dimensional structure that facilitate dCas9 binding, inducing HRP fusion and activation. Reproduced with permission from ( Wang et al, 2020 ). Copyright 2020 American Chemical Society C) Schematic representation of the Cas12a RNP and Cas13a RNP posing target-dependent trans-cleavage activity, resulting in a degradation of linker DNA, inhibiting AuNP aggregation.…”

“…Qiu et al developed a colorimetric detection system comprising of isothermal amplification, detection and reporting based on rolling circle amplification (RCA) in a single reaction tube ( Wang et al, 2020 ). The RCA step allows the dsDNA targeting dCas9 to sense microRNA (miRNA) sequences with single nucleotide precision in clinical serum samples ( Fig.…”

“… 15–60 min ~65 °C ( Li et al, 2019 ; Mukama et al, 2020a ; Qian et al, 2020 ) RCA Rolling circle amplification starts from a circular DNA template and a short DNA or RNA primer to form a long single stranded molecule. ~120 min 20–37 °C Wang et al, 2020 RPA Recombinase polymerase amplification is a low temperature DNA and RNA amplification technique. 5–60 min 37–42 °C ( Y. Chang et al, 2019 ; English et al, 2019 ; Kellner et al, 2019 ; Khan et al, 2019 ; Sullivan et al, 2019 ; X. Wang et al, 2020 ; Williams et al, 2019 ) SDA Strand Displacement Amplification (SDA) employs a restriction endonuclease, which is capable of nicking of its recognition site, and a DNA exonuclease deficient polymerase, which is capable of initiating synthesis at a nick.…”

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

“…It should be noted that the detection limit of CRISPR-Cas-mediated amplification-free nucleic acid detection is usually at the pM level [ 62 ], and recently, several CRISPR-Cas-based biosensors have improved the sensitivity to aM, or even zM, by target amplification [ 74 , 75 ]. In this respect, all the biosensors described here give us rapid, real-time, precise, and authentic information about miRNA molecule detection with desirable sensitivity and specificity.…”

A sensitive and specific method for microRNA detection and in situ imaging based on a CRISPR–Cas9 modified catalytic hairpin assembly

“…According to previous reports, Cas9 requires a 5 0 -NGG-3 0 PAM site to cleave target dsDNA. 23 To further optimize the PAM sequence, we designed different sequences (5 0 -AGG-3 0 , 5 0 -CGG-3 0 , 5 0 -TGG-3 0 , 5 0 -GGG-3 0 and 5 0 -GCC-3 0 ). As shown in Fig.…”