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

Abstract

We report here a method for the molecular detection of miRNAs in exosomes and imaging in living cells based on CRISPR–Cas9 and catalytic hairpin assembly.

Advances in Analytical Technologies for Extracellular Vesicles

“…With a growing focus on EV NAs as markers of the liquid biopsy, various emerging biosensor technologies such as fluorescence-based methods, , SERS-based methods, , electrochemical methods, − the photonic resonator absorption microscopy assay, CRISPR/Cas system-based methods, , etc ., have been developed, providing many opportunities for realizing the POC testing or personalized medicine. Many reviews are focusing on these technologies. − These methods enable efficient, rapid, and robust analysis of EV NAs; however, manual NA extraction step is needed, which may increase the labor intensity when analyzing multiple samples.…”

“…Non-In Situ Methods. With a growing focus on EV NAs as markers of the liquid biopsy, various emerging biosensor technologies such as fluorescence-based methods, 339,340 SERSbased methods, 341,342 electrochemical methods, 343−346 the photonic resonator absorption microscopy assay, 347 CRISPR/Cas system-based methods, 348,349 etc., have been developed, providing many opportunities for realizing the POC testing or personalized medicine. Many reviews are focusing on these technologies.…”

Intracellular Signal Amplification for Ultrasensitive Detection and Imaging: Progress, Challenges, and Opportunities

“…Enzyme-mediated intracellular signal amplification techniques have received extensive attention because of their enhanced amplification performance, excellent sensitivity, and high detection efficiency. Diverse enzymes, such as nicking enzyme, [21,22] duplex-specific nuclease (DSN), [23,24] exonuclease III (Exo III), [25][26][27] DNA polymerase, [11,[28][29][30][31] and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (Cas) systems (CRISPRÀ Cas), [32,33] are used to detect biomolecules and metal ions in living cells for linear, quadratic, and exponential amplification.…”

“…Liu et al constructed a fluorescent sensor by combining CHA amplification with CRISPRÀ Cas9 specific cleavage to realise the in situ detection and imaging of miRNA-21 at a femtomolar level (Figure 3). [32] Under the guidance of a single crRNA, CRISPRÀ Cas12a can recognise dsDNA using a protospacer adjacent motif sequence to form a Cas12aÀ crRNAÀ DNA complex, activating specific ciscleavage activity and nonspecific trans-cleavage of ssDNA. Activated Cas12a can circularly cleave fluorescent reporter substrates, thereby amplifying detectable signals.…”

“…constructed a fluorescent sensor by combining CHA amplification with CRISPR−Cas9 specific cleavage to realise the in situ detection and imaging of miRNA‐21 at a femtomolar level (Figure 3). [32] …”