Abstract
MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.
Highlights
MicroRNAs include a set of short, noncoding proteins and small RNA molecules with a length of 20–24 nt, generated by the RNase-III-type enzyme Dicer from an endogenous transcript that contains a local hairpin structure [1,2]
LbuCas13a: Leptotrichia buccalis Cas13a; LwaCas13a: Leptotrichia wadei Cas13a; AsCas12a: Acidaminococcus sp Cas12a; catalytic hairpin DNA circuit (CHDC): Catalytic hairpin DNA circuit; recombinase polymerase amplification (RPA): Recombinase polymerase amplification; PCR: Polymerase chain reaction; RCA: Rolling circle amplification; catalytic hairpin assembly (CHA): Catalytic hairpin assembly; EXPAR: Isothermal exponential amplification; 1 attomol (10−18 mol); 1femtomolar (1× 10−15 molar); 1 picomolar (1 × 10−18 molar); * According to Zhou et al, the limit of detection can be defined as the target concentration that yields a net signal equivalent to three times the standard deviation of three replicates of the control sample without the target [66]
MiRNAs are becoming the biomarkers in clinical diagnostics of multiple diseases due to their dysregulation associated with many different conditions, such as cancer [68,69], dementia [70], and cardiovascular diseases [71,72]
Summary
MicroRNAs (miRNA) include a set of short, noncoding proteins and small RNA molecules with a length of 20–24 nt, generated by the RNase-III-type enzyme Dicer from an endogenous transcript that contains a local hairpin structure [1,2]. Considering the master role that miRNAs play in regulating critical biological processes and that their ability to repress. Cells 2021, 10, 1655 master role that miRNAs play in regulating critical biological processes and that their ability to repress their targets crucially depends on their expression levels [3,5,11], it is targets their expression levels [3,5,11], it isdetection highly desirable to highly their desirable to crucially constructdepends specific on and ultrasensitive methods for the of construct and ultrasensitive methods for the detection of miRNAs showing low miRNAs showingspecific low expression levels
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