Engineering and optimising deaminase fusions for genome editing

TLDR

Precise editing is essential for biomedical research and gene therapy, yet homology‑directed genome modification is limited by the need for genomic lesions, homology donors, and endogenous DNA repair machinery. The study engineered programmable cytidine deaminases to introduce site‑specific C→T transitions without targeted genomic lesions, aiming to improve processivity and DNA‑binding affinity for safer therapeutic applications. Programmable cytidine deaminases were engineered to catalyze site‑specific C→T transitions in the absence of targeted genomic lesions. Programmable cytidine deaminases achieved 13 % C→T conversion in *E.

Abstract

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.