Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.

Area of Science:

• Molecular Biology
• Genetic Engineering
• RNA Therapeutics

Background:

• Current gene editing technologies in eukaryotic cells are restricted to minor DNA alterations, often causing permanent, unintended genetic modifications.
• Delivering large DNA payloads for gene correction presents significant challenges, limiting therapeutic applications.

Approach:

• We introduce Splice Editing, a versatile platform for *in situ* gene transcript correction.
• This method utilizes CRISPR-mediated RNA targeting combined with the cell's natural RNA-splicing machinery to insert or replace large RNA segments.
• The process achieves precise, large-scale RNA editing without inducing DNA cleavage or mutagenesis.

Key Points:

• Splice Editing enables efficient and specific correction of targeted RNA and protein products, confirmed by RNA sequencing and protein analysis.
• Novel miniature RNA-targeting CRISPR-Cas systems were discovered and characterized for effective delivery to human cells.
• This technology facilitates various editing types across multiple targets and cell lines, editing thousands of bases in a single, reversible step.

Conclusions:

• Splice Editing expands therapeutic possibilities for monogenic diseases with extensive allelic diversity.
• It offers a reversible, *in situ* RNA editing solution, mitigating the risks of permanent unintended effects associated with DNA editing.