SPRINT: a Cas13a-based platform for detection of small molecules

Abstract

Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.

Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli

“…For example, the fluoride riboswitch that controls expression of a downstream fluorophore-binding aptamer was developed into a field-deployable sensor that can rapidly assess fluoride ion concentrations in municipal water supplies with sufficient sensitivity to detect concentrations that exceed acceptable levels by only a few fold [ 91 ]. More recently, a Cas13-based approach has been developed that can covert detection of a small molecule by a riboswitch into a fluorescence output in a single-pot, isothermal reaction in ten minutes [ 92 ]. The bottleneck in extending these applications to a greater variety of small molecule sensors is the development novel biosensors with efficient coupling of an input (the aptamer) to a readily-detectible output.…”

Research on Real-Time Detection of Sprint Error Based on Visual Features and Internet of Things

“…e correct sprint movement technology can reduce the energy consumption of athletes in the process of running. e wrong sprint technique will increase the external resistance, seriously hinder the running speed of athletes, and then affect the performance [2]. Under normal circumstances, the judgment of sprinters' mistakes is mainly based on the use of eye observation by referees or experts.…”

Structure‐Switching RNAs: From Gene Expression Regulation to Small Molecule Detection

“…[ 100 ] An even simpler small molecule‐sensing technology called SPRINT was also recently introduced in which a riboswitch is used to control the transcription of a downstream tag sequence later quantified by the Cas13a‐mediated technology SHERLOCK. [ 101 ] Yet, even though these methods are sensitive, their readout is rather indirect, a limitation that can be overcome by using more direct RNA fluorogenic reporters. No naturally fluorescent RNA molecule has been discovered so far.…”