Programmable RNA writing with trans-splicing

Area of Science:

• Molecular Biology
• Genetic Engineering
• RNA Therapeutics

Background:

• RNA editing offers transient transcriptome modification without permanent off-target effects, but arbitrary edits remain challenging.
• Current RNA editing tools have limitations in scope and efficiency for complex genetic modifications.

Approach:

• Developed Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure (PRECISE), a method utilizing Cas7-11 cleavage to enhance RNA trans-splicing efficiency.
• PRECISE introduces exogenous templates for precise exon replacement or insertion into pre-mRNAs, achieving high editing rates (5-50% endogenous, 85% reporter).

Key Points:

• Demonstrated PRECISE editing across 11 endogenous transcripts, enabling over 50 edit types, including all base transitions/transversions, and insertions/deletions up to 1,863 nucleotides.
• Successfully edited MECP2 for Rett Syndrome, SHANK3 for Autism, and HTT for Huntington's disease, showcasing therapeutic potential.
• Whole transcriptome sequencing confirmed high precision and lack of off-target trans-splicing activity.

Conclusions:

• PRECISE editing broadens genetic editing capabilities, offering a versatile alternative to existing tools like prime editing.
• Achieved protein-free, high-efficiency trans-splicing via payload engineering and ribozymes, with successful AAV delivery for Huntington's disease models.
• PRECISE editing functions in non-dividing cells and addresses a wider spectrum of genetic diseases, including those not treatable by current RNA base editors.