A Chemically Induced CRISPR/dCas13^FCPF Platform for Precise and Programmable RNA Regulation

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Gene Editing

Background:

• Alternative splicing regulates protein diversity but its dysregulation is linked to diseases like cancer and neurodegeneration.
• Current small-molecule splicing modulators lack specificity, causing off-target effects.
• CRISPR/Cas13 systems offer transcript-level targeting but dCas13 fusion effectors can be large and disrupt RNA homeostasis.

Purpose of the Study:

• To develop a precise and controllable RNA modulation system by combining CRISPR targeting with small-molecule action.
• To create a modular platform, Chem-CRISPR/dCas13^FCPF, for targeted splicing modulation with enhanced specificity and reduced dosage.

Main Methods:

• Developed Chem-CRISPR/dCas13^FCPF, a system covalently linking a small molecule to dCas13 via an FCPF π-clamp tag.
• Utilized CRISPR RNA (crRNA) to guide the dRfxCas13d^FCPF effector to the SMN2 exon 7 splice region.
• Assessed splicing modulation by measuring exon inclusion and off-target effects on known sensitive transcripts.

Main Results:

• Chem-CRISPR/dCas13^FCPF induced SMN2 exon 7 inclusion at ligand doses approximately 500-fold lower than free risdiplam.
• The system demonstrated no detectable off-target effects on other risdiplam-sensitive transcripts in the assays.
• The platform showed generalizability to other transcripts through crRNA redesign.

Conclusions:

• Chem-CRISPR/dCas13^FCPF provides a proximity-induced, chemically controllable method for precise RNA modulation.
• This approach couples CRISPR's targeting capability with dose-sparing chemical action for potential therapeutic applications.
• The system represents a significant advancement in developing targeted RNA-based therapies with improved specificity and reduced side effects.