A split cytosine deaminase architecture enables robust inducible base editing

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Base editing technology allows precise genome modification but faces challenges with off-target edits.
• Cytosine to thymine base editors, in particular, raise concerns about genomic instability and tumorigenesis due to unpredictable off-target activity.

Purpose of the Study:

• To develop a controllable base editing tool that minimizes off-target effects.
• To engineer an inducible base editor activated by a clinically safe chemical, rapamycin.

Main Methods:

• Designed four split-human APOBEC3A (A3A)-BE3 base editors by splitting A3A at sites opposite the protein-nucleotide interface.
• Utilized a rapamycin-inducible interaction system (FRB and FKBP) for deaminase reconstruction.
• Tested the editing efficiency and specificity of the split editors in the presence and absence of rapamycin.
• Expanded the strategy to rat APOBEC1 (rA1).

Main Results:

• Three out of four split-A3A-derived base editors demonstrated robust inducible editing upon rapamycin addition.
• Editing activity was significantly inhibited in the absence of rapamycin, confirming inducibility.
• Splicing at Aa85 of A3A yielded the most efficient inducible editing.
• Splitting deaminases did not significantly alter the editing window or motif preference but improved product purity.
• Similar inducible responses were observed with rat APOBEC1.

Conclusions:

• Splitting deaminases is a viable strategy for creating controllable base editing tools.
• This inducible system offers enhanced safety by allowing precise temporal control over base editing activity.
• The developed technology holds promise for safer genome editing applications, potentially overcoming major hurdles for clinical translation.