An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy

Area of Science:

• Molecular Biology
• Gene Editing Technology
• Cancer Research

Background:

• CRISPR/Cas9 is a powerful genome editing tool with broad biomedical potential.
• Safety concerns, particularly off-target effects, limit CRISPR/Cas9 applications.
• Pre-mRNA splicing is a crucial gene expression step in eukaryotes.

Purpose of the Study:

• To develop a novel CRISPR/Cas9 system with enhanced safety and precision.
• To mitigate off-target effects associated with CRISPR/Cas9 gene editing.
• To explore the therapeutic potential of regulated gene editing in cancer.

Main Methods:

• Development of a spliceosome-responsive CRISPR/Cas9 (SpC) editor.
• Utilized pladienolide B (PB) to inhibit splicing and control anti-CRISPR protein expression.
• Validated SpC editor efficacy through in vitro and in vivo bioluminescence imaging.
• Designed a dual-target sgRNA to target the diphtheria toxin A gene in cancer cells.

Main Results:

• The SpC editor precisely regulated Cas9 nuclease activity by controlling anti-CRISPR protein expression.
• In vitro and in vivo studies confirmed the reliability and robustness of the SpC editor.
• Targeting the diphtheria toxin A gene induced apoptosis and inhibited tumor cell growth across various cancer types.

Conclusions:

• The SpC editor offers precise control over gene editing, significantly reducing off-target effects.
• This technology demonstrates potential for targeted cancer gene therapy by regulating tumor cell growth.
• The findings provide new insights into safe and effective CRISPR-based therapeutic strategies.