Type III CRISPR-mediated flexible RNA excision with engineered guide RNAs.

Area of Science:

• Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• Current RNA editing technologies are primarily restricted to single-base modifications.
• CRISPR-based RNA editing typically relies on guide RNA length for cleavage site determination in 6-nucleotide increments.

Purpose of the Study:

• To develop a novel RNA editing system for precise, flexible RNA fragment excision.
• To engineer guide RNAs for CRISPR-Cas systems to enable non-6-nucleotide cleavage and repair.
• To demonstrate the application of this system in correcting mutations and creating novel therapeutic epitopes.

Main Methods:

• Development of the Selective Cleavages and Intramolecular Stitches of RNA (SCISSOR) system.
• Engineering of guide RNAs with specific bulge loop structures to control cleavage sites.
• Systematic evaluation of guide RNA bulge loop lengths (1-24 nt) for precise RNA targeting.
• Demonstration of SCISSOR's efficacy in modifying open reading frames in human cells.

Main Results:

• SCISSOR allows for precise RNA cleavage and ligation at non-6-nucleotide intervals.
• The system accommodates a wide range of bulge loop sizes in guide RNAs, enabling flexible excision.
• SCISSOR successfully repaired frameshift mutations and introduced frameshifts to generate immunogenic poly-epitopes.
• The system demonstrated effectiveness in human cells.

Conclusions:

• SCISSOR provides a versatile platform for targeted RNA manipulation, overcoming limitations of existing methods.
• This technology has significant potential for applications in RNA therapy, including the correction of genetic disorders.
• SCISSOR opens new possibilities for designing therapeutic strategies and advancing biomedical research.