Engineering APOBEC3A deaminase for highly accurate and efficient base editing

Area of Science:

• Molecular Biology
• Genome Editing
• Biochemistry

Background:

• Cytosine base editors (CBEs) enable C-to-T conversions but face limitations due to off-target and bystander effects.
• Existing CBEs exhibit variable efficacy influenced by DNA methylation and sequence context.

Purpose of the Study:

• To engineer highly accurate and efficient adenine base editors (haA3A-CBEs) with reduced off-target activity.
• To assess the therapeutic potential of haA3A-CBEs in a mouse model of tyrosinemia.

Main Methods:

• Structure-guided engineering of human APOBEC3A (A3A) deaminase to create novel haA3A-CBE variants.
• Compatibility testing with PAM-relaxed SpCas9-NG for precise targeting of pathogenic mutations.
• In vivo delivery using adeno-associated virus (AAV) and lipid nanoparticle (LNP)-based mRNA in a mouse model.

Main Results:

• Developed haA3A-CBE variants with a narrow editing window and near-background off-target DNA and RNA activity.
• Achieved up to 58.1% editing efficiency in mouse liver tissue for tyrosinemia, with minimal bystander editing.
• LNP-based mRNA delivery further reduced off-target effects compared to AAV delivery.

Conclusions:

• Engineered haA3A-CBEs offer enhanced precision and efficiency for C-to-T base editing.
• These advanced CBEs demonstrate significant potential for developing novel therapies for genetic diseases.
• The developed system overcomes limitations of previous base editors, paving the way for clinical applications.