As aC RISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here,w ed escribe an unreported capacity of Cas14a1:R NA can trigger the trans ssDNAc leavage.T his Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNAc leavage,A TCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNAactivation. Using ATCas-RNAv ia af luorophore quencherlabeled ssDNAr eporter (FQ), we were able to detect 1aM pathogenic nucleic acid within 1h,and achieve 100 %accuracy with 25 milk samples.This platform can serve as anew tool for high-efficiency nucleic acid diagnostics.Importantly,this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.
Nanopore
technology is promising for the next-generation of nucleic
acid–based diagnosis. However, sequence reservation could still
be hardly achieved in low-concentration. Herein, we propose a trypsin-activated
catalysis reaction for amplified detection, which substantially improves
the sensitivity of nanopore technique. The proposed trypsin-amplified
nanopore amplified sandwich assay (tNASA) could contribute to a sensitivity
approximately 100 000 times higher based on nucleic acid probe
design. Remarkably, tNASA is capable of attomolar nucleic acid and
single cell detection by using a miniaturized current amplifier without
alignment algorithm. Also it allows 10 pathogenic species in serum
to be accurately and robustly profiled, thus be utilized for the diagnosis
of infectious diseases. tNASA may evolve the construction of nanopore
techniques for nucleic acid detection and would facilitate its translation
for pocket diagnosis and precision medicine.
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