Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Noncoding RNAs (ncRNAs) are crucial biological molecules with diverse functions.
• Harnessing the capabilities of ncRNAs requires effective methods for their targeted delivery and study.
• Existing technologies may limit the size and complexity of RNA cargos that can be deployed.

Purpose of the Study:

• To develop a novel method for targeted localization of large RNA cargos to specific DNA loci.
• To enable the investigation and manipulation of noncoding RNA functions.
• To create a versatile platform for deploying functional RNA domains and ribonucleoprotein complexes.

Main Methods:

• Development of clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp).
• Utilizing Cas9 nuclease for targeted DNA binding and RNA cargo delivery.
• Insertion of functional RNA domains (up to 4.8 kb) into CRISPR guide RNA.

Main Results:

• Demonstrated successful insertion of large functional RNA domains into CRISPR guide RNA.
• Constructed Cas9 complexes with diverse RNA cargos, including protein-binding cassettes, aptamers, random sequences, and natural long noncoding RNAs.
• Showcased the multiplexing capability of CRISP-Disp for targeting distinct functions to multiple genomic sites.

Conclusions:

• CRISP-Disp is a powerful and versatile tool for targeted RNA localization.
• The method allows for the deployment of large and complex RNA molecules to specific genomic locations.
• CRISP-Disp opens new avenues for functional RNA research and biotechnological applications.