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.
Subject(s)
Bacterial Proteins/metabolism , Biosensing Techniques/methods , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Enzyme Assays/methods , Nucleic Acid Synthesis Inhibitors/analysis , Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Endodeoxyribonucleases/genetics , Fluorescent Dyes/chemistry , Leptotrichia , Ligands , Nucleic Acid Synthesis Inhibitors/pharmacology , Riboswitch , Rifampin/analysis , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.
Subject(s)
CRISPR-Cas Systems , Escherichia coli/genetics , Gene Editing/methods , Cloning, Molecular , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Expression Regulation , Nucleic Acid Conformation , RNA, Guide, Kinetoplastida , TheophyllineABSTRACT
Sensory photoreceptors evoke numerous adaptive responses in nature and serve as light-gated actuators in optogenetics to enable the spatiotemporally precise, reversible, and noninvasive control of cellular events. The output of optogenetic circuits can often be dialed in by varying illumination quality, quantity, and duration. A programmable matrix of light-emitting diodes has been devised to efficiently probe the response of optogenetic systems to intermittently applied light of varying intensity and pulse frequency. Circuits for light-regulated gene expression markedly differed in their responses to pulsed illumination of a single color which sufficed for their sequential triggering. In addition to quantity and quality, the pulse frequency of intermittent light hence provides a further input variable for output control in optogenetics and photobiology. Pulsed illumination schemes allow the reduction of overall light dose and facilitate the multiplexing of several lightdependent actuators and reporters.
Subject(s)
Lighting/methods , Optogenetics/methods , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation/radiation effects , Histidine Kinase/genetics , Kinetics , Light , Photoreceptors, Microbial/geneticsABSTRACT
The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.
