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

Area of Science:

• Biological Engineering
• Molecular Biology
• Biotechnology

Background:

• CRISPR-based methods offer rapid, sensitive, and cost-effective nucleic acid detection.
• Cas13a enzymes are programmable RNA-guided ribonucleases with potential beyond nucleic acid detection.

Purpose of the Study:

• To adapt a Cas13a-based system for the detection of small molecules in a one-batch assay.
• To develop a method for quantifying various compounds using a coupled transcriptional reporter system.

Main Methods:

• SHERLOCK-based profiling of in vitro transcription (SPRINT) utilizes Cas13a's RNase activity triggered by in vitro transcribed RNA.
• A riboswitch or protein-based regulatory system couples effector molecule concentration to transcriptional output.
• Cleavage of a quenched fluorophore-labeled RNA by activated Cas13a generates a measurable fluorescence signal.

Main Results:

• Quantified eight diverse compounds, including cofactors, nucleotides, amino acid metabolites, tetracycline, and monatomic ions.
• Achieved high-throughput quantification of hundreds of reactions within hours.
• Demonstrated the adaptability of SPRINT for characterizing transcriptional regulators and inhibitors.

Conclusions:

• SPRINT provides a versatile and sensitive platform for small molecule detection using CRISPR-Cas13a technology.
• The assay's adaptability to portable formats enables potential field and point-of-care applications.
• This method expands the utility of Cas13a systems for diverse biological and chemical sensing applications.