Engineering RsDddA as mitochondrial base editor with wide target compatibility and enhanced activity

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Double-stranded DNA-specific cytidine deaminase (DddA) base editors are valuable tools in biomedical research, medicine, and biotechnology.
• A significant limitation of current DddA editors is their strict sequence preference, hindering their broader application.
• Overcoming sequence-context constraints is crucial for maximizing the potential of DddA base editors.

Purpose of the Study:

• To identify and engineer a novel DddA homolog with improved sequence compatibility for base editing.
• To develop and characterize a DddA-derived cytosine base editor (RsDdCBE) for efficient mitochondrial base editing.
• To assess the editing efficiency and off-target effects of the engineered RsDdCBE in mammalian cells.

Main Methods:

• Bioinformatic analysis of protein datasets to identify novel DddA homologs.
• Engineering of a DddA homolog from *Ruminococcus* sp. *AF17-6* (RsDddA) for mitochondrial targeting.
• Demonstration of base editing in a mammalian cell line and analysis of editing efficiency and off-target frequencies.

Main Results:

• Identification and engineering of a novel DddA homolog, RsDddA, from *Ruminococcus* sp. *AF17-6*.
• RsDddA-derived cytosine base editors (RsDdCBE) exhibit broadened NC sequence compatibility.
• RsDdCBE demonstrates robust editing efficiency in mammalian cells with off-target editing frequencies comparable to existing DdCBE variants.

Conclusions:

• The engineered RsDddA provides a promising alternative to overcome sequence-context limitations of DddA base editors.
• RsDdCBE expands the utility of DddA base editors for mitochondrial genome editing applications.
• The developed RsDdCBE offers a powerful tool for biomedical research, medicine, and biotechnology with improved specificity.