CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Synthetic Biology

Background:

• CRISPR technologies offer powerful tools for gene function studies and manipulation.
• RNA aptamers fused to CRISPR's single-guide RNA (sgRNA) can recruit RNA-binding proteins (RBPs) to specific genomic locations.
• Multiplexed gene regulation is achievable by expressing sgRNAs with various RNA aptamers.

Purpose of the Study:

• To develop an intracellular directed evolution platform for RNA aptamers targeting intracellularly expressed RBPs.
• To identify high-affinity RNA aptamers that are orthogonal to existing aptamer-RBP pairs.
• To demonstrate the application of these orthogonal aptamer-RBP pairs in multiplexed CRISPR systems for simultaneous gene regulation in mammalian cells.

Main Methods:

• Optimization of a bacterial CRISPR-hybrid system integrated with Fluorescence-Activated Cell Sorting (FACS).
• Intracellular directed evolution to select for high-affinity RNA aptamers.
• Validation of aptamer orthogonality against known aptamer-RBP interactions.
• Application in multiplexed CRISPR systems for transcriptional activation and repression.

Main Results:

• Successful identification of high-affinity RNA aptamers through the directed evolution platform.
• Demonstrated orthogonality of the newly identified aptamers to existing aptamer-RBP pairs.
• Effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells using multiplexed CRISPR with orthogonal aptamer-RBP pairs.

Conclusions:

• The developed intracellular directed evolution platform is effective for generating orthogonal RNA aptamers.
• Multiplexed CRISPR systems utilizing orthogonal aptamer-RBP pairs enable precise and simultaneous control of multiple endogenous genes.
• This technology advances the capabilities of CRISPR-based gene regulation for research and potential therapeutic applications.