CRISPR/Cas13 sgRNA-Mediated RNA-RNA Interaction Mapping in Live Cells with APOBEC RNA Editing

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• Long non-coding RNAs (lncRNAs) primarily studied for protein interactions and genomic binding.
• RNA-RNA interactions mediated by lncRNAs remain largely unexplored.
• Existing methods lack the specificity to comprehensively map lncRNA-RNA interactomes.

Purpose of the Study:

• To develop and validate a novel method for identifying RNA partners of lncRNAs.
• To investigate the lncRNA-RNA interactome using the new method.
• To expand the understanding of lncRNA functions in gene regulation.

Main Methods:

• Developed sarID (sgRNA scaffold assisted RNA-RNA interaction detection).
• Integrated Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing.
• Applied sarID to lncRNAs NEAT1, XIST, MALAT1, NBR2, and DANCR.

Main Results:

• Discovered over one thousand previously unidentified binding transcripts for NEAT1.
• Demonstrated broad applicability of sarID across multiple lncRNAs.
• Identified novel RNA-RNA interactions, suggesting new regulatory roles for lncRNAs.

Conclusions:

• lncRNAs may regulate gene expression through interactions with messenger RNAs (mRNAs).
• sarID offers a novel, immunoprecipitation-free approach for RNA interactome studies.
• The findings expand the known functional repertoire of lncRNAs beyond protein scaffolding or miRNA sponging.