A split and inducible adenine base editor for precise in vivo base editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• DNA base editors enable targeted nucleotide conversions.
• Existing adenine base editors (ABE) using TadA deaminases lack cellular control.
• Post-translational control is crucial for precise gene editing applications.

Purpose of the Study:

• To develop a split adenine base editor (sABE) with chemically induced dimerization (CID) for controlled DNA editing.
• To assess the activity, precision, and off-target effects of sABE compared to conventional ABE.
• To demonstrate the in vivo applicability of CID-controlled base editing.

Main Methods:

• Constructing a split adenine base editor (sABE) system utilizing chemically induced dimerization (CID).
• Evaluating sABE's on-target editing efficiency and background activity in cellular models.
• Assessing genomic and transcriptomic off-target mutations and editing precision (e.g., single-to-double ratio).
• Implementing sABE for gene knockout via multiplex splice donor disruption.
• Delivering sABE via adeno-associated virus (AAV) vectors for in vivo editing in mouse models.

Main Results:

• sABE demonstrated high on-target editing activity comparable to ABE8e upon rapamycin induction.
• sABE exhibited significantly lower background activity without induction.
• sABE showed enhanced precision with a narrower activity window and improved single-to-double adenine editing ratio.
• Reduced genomic and transcriptomic off-target effects were observed with sABE.
• Successful gene knockout and in vivo A•T to G•C base conversion in the PCSK9 gene in mouse liver were achieved.

Conclusions:

• sABE provides precise, inducible control over DNA base editing activity.
• The enhanced precision and reduced off-target effects of sABE are advantageous for therapeutic applications.
• CID-controlled base editing using sABE holds significant promise for both basic research and in vivo gene therapy.