TALE base editors (TALEB) are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves coupled to an uracil glycosylase inhibitor (UGI). By converting a cytosine (C) to a thymine (T) via the formation of an uracil intermediate, TALEB function as a C-to-T class of base editor able to edit double strand DNA.

Taking advantage of a highly precise and efficient TALEN®-mediated ssODN knock-in in primary T cells, we applied a strategy that allowed the comprehensive characterization of C-to-T conversion efficiencies, how target composition, and spacer variations, affect TALEB activity/efficiency within the editing window.

Notably, we highlighted that the composition of bases surrounding the target TC may strongly influence the editing efficiencies. We also demonstrated that by varying the TALEB scaffold, we could not only relax target sequence limitations, but with thoughtful consideration modulate bystander editing within the editing window, overall allowing TALEB to be fine-tuned for a desired gene editing outcome.

We then thought to further assess characteristics of nuclear genome editing by applying a range of different techniques. First focusing on on-target editing, we also explored the possibility, and risk, associated with genome wide TALE dependent/independent binding and editing. Using an experimental model, we demonstrated that a single TALEB arm does not lead to detectable editing (detection limit: 0.1-0.2%) which we then validated with hybrid capture assays.

Altogether, the datasets obtained in this study enhanced our understanding of TALEB and permits the design of extremely efficient and specific TALEB, compatible with the development of future therapeutic applications.