Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.

Area of Science:

• Molecular Biology
• Genetic Engineering
• RNA Therapeutics

Background:

• CRISPR-Cas systems offer powerful genetic editing capabilities.
• Current CRISPR systems lack precise RNA-dependent activation mechanisms.
• Targeting specific RNA molecules for genetic modulation remains a challenge.

Purpose of the Study:

• To engineer novel CRISPR guide RNAs (igRNAs) for RNA-dependent activation.
• To develop customizable CRISPR systems responsive to specific RNA molecules.
• To enhance specificity and orthogonality in CRISPR-based genetic modulation.

Main Methods:

• Design and synthesis of cis-repressed RNA-interacting guide RNAs (igRNAs).
• Testing of igRNAs in living *Escherichia coli* cells.
• Validation using cell-free assays for rapid prototyping.

Main Results:

• Demonstrated RNA-dependent activation of CRISPR-Cas systems by igRNAs.
• Successfully triggered CRISPR activity upon interaction with small RNA fragments and relevant mRNA transcripts (e.g., HIV).
• Achieved orthogonal activation and increased specificity in genetic modulation.

Conclusions:

• Novel igRNAs enable RNA-responsive CRISPR-based genetic editing.
• The developed CRISPR-igRNAs are adaptable to various CRISPR-Cas variants.
• Significant potential for therapeutic, diagnostic, and biotechnological applications.