Yang L, Briggs AW, Chew WL, Mali P, Guell M, Aach J, Goodman DB, Cox D, Kan Y, Lesha E, Soundararajan V, Zhang F, Church G (2016) Engineering and optimising deaminase fusions for genome editing. Nature Communications 7:13330. https://doi.org/10.1038/ncomms13330.
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. The research team engineered programmable cytidine deaminases and tested the introduction of site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. The programmable deaminases effectively converted 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.
The study used prepGEM to genotype the GFP target locus in HEK293 cells and demonstrated that engineered deaminases are capable of efficient sequence-specific genome editing in HEK293FT cells.
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