FINDER: A Fluidly Confined CRISPR‐Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification

Abstract

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the natural confined enzyme on fluidic cell membrane, afluidically confined CRISPR‐based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR‐Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamers‐based DNA logic gate for cell recognition, thus a FINDER system with accurate, rapid and sensitive cancer cell identification was constructed. The FINDER can rapidly identified target cells in only 20 min, and achieved over 80% recognition efficiency with only 0.1% of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

Smart DNA sensors‐based molecular identification for cancer subtyping

“…(d) Illustration of fluidly confined CRISPR-based DNA reporter (FINDER) for cancer cell identification. [34] Graph copyright from reference [30][31][32][33][34] .…”

“…In the above examples, benefiting from the advantages of high throughput, precision, and fast speed, flow cytometry (FCM) is the most used technology to determine the heterogeneous characteristics of a single cell depending on fluorescently labeled molecules. [32][33][34] To further perform single-cell analysis for low-abundance target cells, the droplet microfluidic platform is more attractive. Kelly's group [38] reported a fluorescent drop cytometry (FDC) strategy for livecell phenotypic profiling with a small (<40 cells) number of cells (Figure 5a).…”

“…Recently, to realize more rapid and sensitive identification for target cells, our group developed a CRISPR‐based DNA reporter based on fluidly confined cell membranes named FINDER, in which an aptamer‐based DNA logic gate was used for identifying the target cell and CRISPR Cas12a ‐based was used for the signal output (Figure 3d). [34] The activity of CRISPR nuclease was significantly enhanced on the cell membrane due to the confinement effect and membrane fluidity. Finally, the FINDER could identify 0.1% of target cancer cells with over 80% recognition efficiency in 20 min.…”

“…(c) System design and operational mechanism based on HCR [32] and RCA [33] for accurate cancer cell identification. (d) Illustration of fluidly confined CRISPR‐based DNA reporter (FINDER) for cancer cell identification [34] . Graph copyright from reference [30‐34] .…”

DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing

“…The clustered regularly interspaced short palindromic repeats (CRISPR) system originated from bacteria and archaea can provide a prokaryotic adaptive immune defense system to degrade intruding nucleic acids . Owing to the increasing number of scientists eager to investigate and analyze the workings of the CRISPR system in depth, it has recently become a revolutionary tool for gene editing, transcriptional regulation, and molecular diagnostics. − Among the CRISPR-associated proteins (Cas) family, Cas12a is one of the most influential RNA-programming endonucleases that can only exert its target recognition and cleavage activity under the guidance of CRISPR RNA (crRNA) . The crRNA used for Cas12a effector consists of two fragments, a hairpin “scaffold” motif that can bind to the Cas12a protein and is identical in different crRNAs, and a variable “spacer” motif (20 nucleotides, 20 nt) that complements the “protospacer” sequences in the target DNA (activator).…”