Multivalent Duplexed-Aptamer Networks Regulated a CRISPR-Cas12a System for Circulating Tumor Cell Detection

Abstract

Although circulating tumor cells (CTCs) have great potential to act as the mini-invasive liquid biopsy cancer biomarker, a rapid and sensitive CTC detection method remains lacking. CRISPR-Cas12a has recently emerged as a promising tool in biosensing applications with the characteristic of fast detection, easy operation, and high sensitivity. Herein, we reported a CRISPR-Cas12a-based CTC detection sensor that is regulated by the multivalent duplexed-aptamer networks (MDANs). MDANs were synthesized on a magnetic bead surface by rolling circle amplification (RCA), which contain multiple duplexed-aptamer units that allow structure switching induced by cell-binding events. The presence of target cells can trigger the release of free "activator DNA" from the MDANs structure to activate the downstream CRISPR-Cas12a for signal amplification. Furthermore, the 3D DNA network formed by RCA products also provided significantly higher sensitivity than the monovalent aptamer. As a proof-of-concept study, we chose the most widely used sgc8 aptamer that specifically recognizes CCRF-CEM cells to validate the proposed approach. The MDANs-Cas12a system could afford a simple and fast CTC detection workflow with a detection limit of 26 cells mL −1 . We also demonstrated that the MDANs-Cas12a could directly detect the CTCs in human blood samples, indicating a great potential of the MDANs-Cas12a in clinical CTC-based liquid biopsy.

Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids

“…16,17 The diagnostic technologies based on the CRISPR-Cas system have been extensively studied since the trans-cleavage ability of Cas12a indiscriminately for collateral single-strand DNA (ssDNA) was discovered in 2018. 11,18,19 The trans-cleavage ability of Cas12a is the key to the derived biosensing technologies. With up to 1250 turnovers per second trans-cleavage activity, the CRISPR-Cas12a based biosensing platform possesses an ideal self-amplification effect.…”

Towards Point of Care CRISPR-Based Diagnostics: From Method to Device

“…The adenosine 5′-triphosphate (ATP) and sodium ions (Na + ) can be detected at ambient temperature (25 °C) using this method, with a detection limit of 4.75 μM and 0.10 mM. This strategy expanded the detection objects of CRISPR/Cas-based diagnosis to pathogens, cells, exosomes, metal ions, small molecule compounds, and so on, accelerating the development of the CRISPR/Cas-based POCT [ 70 , 71 , 72 , 73 ]. Moreover, when a T7 promoter is added to the functional DNA, the in vitro transcription reaction response begins only after the functional DNA has been unlocked by the analytes [ 74 ].…”

New Insights for Biosensing: Lessons from Microbial Defense Systems

“…Thus, Cas12a is usually more specific than Cas9 in genome editing. Cas 12a uses a single RuvC domain to catalyze the cleavage of cis -/ trans -ssDNA or dsDNA guided by RNA. − In contrast, Cas9 utilizes two catalytic (RuvC and HNH) domains for RNA-guided dsDNA cleavage …”