Highly Effective Detection of Exosomal miRNAs in Plasma Using Liposome-Mediated Transfection CRISPR/Cas13a

Abstract

Exosomal miRNAs play a critical role in cancer biology and could be potential biomarkers for cancer diagnosis. However, due to the low abundance of miRNAs in the exosomes, recognizing and detecting disease-associated exosomal miRNAs in an easy-to-operate way remain a challenge. Herein, we used a liposome-mediated membrane fusion strategy (MFS) to transfect CRISPR/Cas13a into exosomes, termed MFS-CRISPR, directly measuring exosomal miRNAs in plasma. Using the MFS-CRISPR platform for detection of the exosomal miR-21, we achieve a linear range spanning four orders of magnitude (104–108 particles/mL) and the method is able to detect the exosomal miR-21 in as low as 1.2 × 103 particles/mL. The liposome-mediated MFS could confine fluorescent signals in fused vesicles, which can be used for exosome heterogeneity analysis. Moreover, MFS-CRISPR assay was evaluated by measuring clinical samples, and the difference of miR-21 expression of breast cancer patients and healthy donors was significant. Because of high sensitivity and simplicity, the proposed method could have promising clinical potential for cancer diagnosis and treatment monitoring.

CRISPR/Cas detection with nanodevices: moving deeper into liquid biopsy

“…Similarly, Zhang and coworkers employed a liposome-mediated membrane fusion strategy (MFS) for CRISPR/Cas13a transfection to detect exosomal miRNAs in plasma. 72 This MFC-CRISPR platform resulted in the effective detection of exosomal miR-21 in clinical samples from breast cancer patients, showcasing the promising potential for diagnosis. Such MFS has great potential for in situ liquid biopsy since the emerging membrane-fusogenic liposomes can deliver interested cargos in lipid-coating cells and EVs via fusion-mediated uptake.…”

“…Furthermore, nanoliposome-transfected platforms have been applied for the detection of exosomal miRNA. 71,72 In a recent innovation, a liposome-transfected CRISPR/Cas12a platform was developed to detect SARS-CoV-2 RNA-positive extracellular vesicles in plasma (Fig. 9B).…”

Application of CRISPR/Cas13a-based biosensors in serum marker detection

“…It uses the trans cleavage of Cas13a to release the target of Cas12a, thereby triggering the cleavage activity of Cas12a to produce a uorescent signal with a detection limit of 0.35 fM. Zhang et al 47 established a biosensor for MFS-CRISPR by transfecting Cas13a into extracellular vesicles using a liposome-mediated membrane fusion strategy (MFS), which directly measured extracellular miRNA in plasma. They encapsulated the Cas13a detection system (Lwa-Cas13a, crRNA, and a uorescent probe) in liposomes and delivered the system to exosomes by membrane fusion (Fig.…”

Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA

“…The gold standard for miRNAs assay is quantitative reverse transcription polymerase chain reaction (qRT-PCR). 11,12 However, this approach needs complementary DNA (cDNA) synthesis, the sophisticated design of primers and tremendous amounts of time. 13,14 Recently, various PCRfree analytical approaches have been developed for exosomal miRNAs determination, for example, Ding's group developed a surface plasmon resonance imaging (SPR) platform to detect exosomal miRNAs based on the silver nanocube.…”

“…Among them, exosomal miRNAs as biomarkers have attracted a great deal of attention for cancer diagnosis. The gold standard for miRNAs assay is quantitative reverse transcription polymerase chain reaction (qRT-PCR). , However, this approach needs complementary DNA (cDNA) synthesis, the sophisticated design of primers and tremendous amounts of time. , Recently, various PCR-free analytical approaches have been developed for exosomal miRNAs determination, for example, Ding’s group developed a surface plasmon resonance imaging (SPR) platform to detect exosomal miRNAs based on the silver nanocube . Fu’s group developed a THz metamaterial biosensor based on duplex-specific nuclease-triggered rolling circle amplification system for exosomal miRNAs assays .…”