Improvement of C-to-U RNA editing using an artificial MS2-APOBEC system

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Editing

Background:

• RNA cytidine deamination, specifically C-to-U editing, is a crucial post-transcriptional modification.
• The MS2-apolipoprotein B-editing catalytic polypeptide-like (APOBEC)1 system has previously enabled RNA editing.
• APOBEC3A and APOBEC3G are cytidine deaminase (CDA) enzymes with known single-stranded RNA preferences.

Purpose of the Study:

• To investigate the RNA editing efficiencies of APOBEC3A and APOBEC3G fused with the MS2 system.
• To design guide RNAs that facilitate single-stranded RNA structures for APOBEC3A and APOBEC3G.
• To enhance APOBEC3G editing efficiency by altering its base preference through site-specific mutation.

Main Methods:

• Fusion of APOBEC3A and APOBEC3G enzymes with the MS2 system.
• Design of unconventional guide RNAs to induce target sequence loops, creating single-stranded RNA structures.
• Introduction of the D317W mutation into APOBEC3G to modify its base preference.
• Co-transfection of HEK 293T cells with MS2-fused enzymes and guide RNAs.
• Assessment of RNA editing efficiencies.

Main Results:

• MS2-fused APOBEC3A and APOBEC3G demonstrated high C-to-U RNA editing efficiency when co-transfected with a loop-inducing guide RNA.
• The D317W mutation in APOBEC3G showed a slight, though not statistically significant, improvement in editing efficiency.
• APOBEC3A and APOBEC3G successfully induced C-to-U RNA editing in the presence of the designed guide RNA.

Conclusions:

• APOBEC3A and APOBEC3G can be utilized for C-to-U RNA editing via the MS2 system with loop guide RNAs.
• Site-specific mutation can enhance the editing efficiency of APOBEC3G by altering its base preference.
• The MS2 system's versatility in fusing with different enzymes suggests broad potential for RNA editing applications.