Compact RNA editors with natural miniature Cas13j nucleases

Area of Science:

• Molecular Biology
• Gene Editing
• Biotechnology

Background:

• CRISPR-Cas13 systems are valuable for RNA editing.
• Large effector sizes limit adeno-associated virus (AAV) delivery for RNA editing therapies.
• Development of smaller, efficient RNA editing tools is crucial for therapeutic applications.

Purpose of the Study:

• To identify and characterize novel, miniaturized CRISPR-Cas13 effectors for RNA editing.
• To develop compact RNA base editors compatible with single-AAV packaging.
• To demonstrate the therapeutic potential of these systems in correcting disease-causing mutations.

Main Methods:

• Identification and characterization of the Cas13j family, including LepCas13j and ChiCas13j.
• Construction of a compact RNA base editor (Chi-RESCUE-S) by fusing dChiCas13j with hADAR2dd.
• In vitro and in vivo validation of RNA editing efficiency and specificity.
• Delivery of the editing system using single-AAV vectors.

Main Results:

• The Cas13j family, particularly ChiCas13j (424 aa), represents the smallest and highly efficient RNA interference variants.
• Chi-RESCUE-S achieved efficient and specific A-to-G and C-to-U RNA base editing.
• The system enabled single-AAV packaging and corrected pathogenic mutations (APOC3^D65N, SCN9A^R896Q).
• In vivo C-to-U editing of PCSK9 in mice using Chi-RESCUE-S-mini3 reduced cholesterol levels.

Conclusions:

• The Cas13j family provides compact and efficient RNA editing tools.
• Miniaturized Cas13j-based base editors are compatible with single-AAV delivery for therapeutic applications.
• These systems show promise for treating genetic diseases through in vivo RNA editing.