Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs

Abstract

CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence which activates Cas enzymes to cleave reporter molecules. Currently, most CRISPRbased diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here, we show the combination of a CRISPR/Cas-based reaction with a Nanozyme-Linked ImmunoSorbent Assay which allows for the quantitative and colorimetric readout of Cas13mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes, and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilise this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme has significant potential as a universally applicable signal catalyst for CRISPR-based diagnostics which will expand the spectrum of targets for preamplification-free, quantitative detection.

Lateral flow test engineering and lessons learned from COVID-19

“…CRISPR-Dx could extend beyond COVID-19 LFT diagnosis and may allow rapid diagnosis of diverse diseases and variant or resistance monitoring. In particular, CRISPR-Dx benefit from high sensitivity in diverse clinical samples 110,119,128,129 , streamlined 'one-pot' protocols and freeze-dried, cell-free assay formats for usability and stability [121][122][123]127,130,131 , as well as smartphone-integrated result interpretation contributing to digital surveillance programmes 122,127,132 . CRISPR-Dx have initially required laboratory equipment for amplification and readout; however, these platforms can also operate with battery power or without power at room temperature 123,125,127 .…”

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

“…Similarly, Marta Broto et al have studied the combination of a CRISPR/Cas-based reaction with a nanozyme-linked immunosorbent assay (CrisprZyme), allowing for the quantitative and colorimetric readout of Cas13-mediated RNA detection. 117 In principle, nanozymes have a higher substrate conversion rate than HRP. 118 However, CrisprZyme has failed to show much improvement on the sensitivity compared with HRP-coupled CRISPR/Cas detection platform, probably because short target sequence restricted crRNA and non-specific immunosorbent.…”

“…118 However, CrisprZyme has failed to show much improvement on the sensitivity compared with HRP-coupled CRISPR/Cas detection platform, probably because short target sequence restricted crRNA and non-specific immunosorbent. 117,118…”

“…118 However, Crispr-Zyme has failed to show much improvement on the sensitivity compared with HRP-coupled CRISPR/Cas detection platform, probably because short target sequence restricted crRNA and non-specific immunosorbent. 117,118 Briefly, the protease-based cascade signal amplification systems have proved their capability of improving sensitivity, but they still require for further optimization to achieve compatible multi-reaction and real clinical applications.…”

CRISPR/Cas Systems‐Inspired Nano/Biosensors for Detecting Infectious Viruses and Pathogenic Bacteria

“…[58,64,[89][90][91][92] Intriguingly, CRISPR/Cas systems have been introduced into the enzyme/ nanozyme-catalyzed colorimetric sensing for the construction of a novel detection platform. [93][94][95][96][97][98][99] As reported by Zhu's group, CRISPR/dCas9 system, split-horseradish peroxidase (HRP) techniques, isothermal amplification, and rolling circle amplification (RCA) were used to establish a new and lowcost RCA-CRISPR-split-HRP (RCH) strategy for the highly efficient detection of miRNAs with a single-base specificity. In this method, the miRNAs could be isothermally amplified by employing the RCA as a primary amplifier to produce the large DNA fragments with the regular stem-loop structures and repeated miRNA complementary sequences.…”