sgRNA amount is a limiting factor in adenine base editing using RNA LNPs

Area of Science:

• Biotechnology
• Genetics
• Molecular Biology

Background:

• Base editors offer precise genome editing with therapeutic potential.
• Optimizing single-guide RNA (sgRNA) delivery and dosage is crucial for maximizing on-target editing and minimizing off-target effects.

Purpose of the Study:

• To investigate the impact of sgRNA dosage on in vivo editing efficiency and tissue specificity.
• To evaluate the relationship between sgRNA dose and off-target/bystander effects in adenine base editing.

Main Methods:

• Utilized a reporter mouse model with a luciferase transgene correctable by adenine base editing.
• Administered lipid nanoparticles co-encapsulating ABE8e RNA and varying sgRNA doses (1 mg/kg and 4 mg/kg).
• Assessed editing outcomes via whole-body imaging, ex vivo tissue analysis, and sequencing.

Main Results:

• Increasing sgRNA dose to 4 mg/kg enhanced editing efficiency up to 3.3-fold across multiple tissues.
• The liver showed the highest on-target editing rates (63%) at the higher sgRNA dose.
• Bystander editing increased significantly and dose-dependently, especially in highly edited tissues like the liver.

Conclusions:

• sgRNA dose optimization is critical for balancing editing efficiency, tissue specificity, and bystander effects in ABE8e applications.
• Higher sgRNA doses enhance therapeutic editing but necessitate careful management of unintended edits.