Engineering and optimising deaminase fusions for genome editing.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Homology-directed genome modification requires specific DNA lesions, homology donors, and cellular repair mechanisms.
• Existing gene editing tools have limitations hindering broad application in research and therapy.

Purpose of the Study:

• To engineer programmable cytidine deaminases for site-specific cytidine to thymidine transitions.
• To assess the efficiency and specificity of these deaminases in prokaryotic and eukaryotic cells.
• To evaluate potential off-target effects and chromosomal abnormalities post-editing.

Main Methods:

• Engineering of programmable cytidine deaminases.
• Testing deaminase efficiency in *Escherichia coli* and human cells.
• Whole genome sequencing to detect off-target mutations and chromosomal integrity.

Main Results:

• Programmable deaminases achieved 13% efficiency in *E. coli* and 2.5% in human cells.
• Off-target deaminations were observed up to 150 bp from the target site.
• Edited bacterial cells showed no chromosomal abnormalities but increased global deamination at binding sites.

Conclusions:

• Programmable deaminases show promise as a novel genome editing tool for prokaryotes and eukaryotes.
• Further engineering is needed to enhance specificity and reduce off-target effects for therapeutic applications.
• Addressing deaminase processivity and DNA binding affinity is crucial for safer genome editing.