Sensing and perturbing mammalian cell states with reprogrammable ADAR sensors (RADARS)

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genetics

Background:

• Adenosine Deaminase Acting on RNA (ADAR) enzymes play crucial roles in RNA editing.
• Targeting specific cell types based on transcriptional signatures is essential for biological research and therapeutics.
• Existing tools for cell-type targeting often lack programmability and adaptability.

Purpose of the Study:

• To introduce Reprogrammable Adenosine Deaminase Acting on RNA (ADAR) Sensors (RADARS) as a novel technology for controlling RNA translation.
• To enable noninvasive sensing or perturbation of specific mammalian cell types.
• To provide a detailed protocol for designing, cloning, and validating RADARS constructs.

Main Methods:

• RADARS utilize base-pairing between a target RNA and a sensor RNA to recruit ADAR.
• ADAR edits a premature stop codon upstream of a gene of interest, releasing translation of a desired cargo.
• RADARS guide sequences are designed using a web interface and cloned into constructs for various applications.

Main Results:

• RADARS demonstrate sequence programmability, allowing adaptation to new contexts.
• The protocol facilitates the design and validation of RADARS constructs for specific transcripts.
• Recommendations for cargo choice, sensor design, and ADAR system selection are provided.

Conclusions:

• RADARS offer a versatile and adaptable platform for cell-specific RNA targeting.
• The technology simplifies the design and execution of cell-targeting experiments.
• RADARS enable advanced applications in imaging, cell sorting, and sequencing.