Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs

“…When using αDdCBEs, we suggest defining spacers ranging from 11 to 18 bp long (preferably up to 16 bp), delimited by TALEs containing 15 or 16 repeats, irrespective of the 5'-most nucleotides of their target sequences. Regarding deaminase domain selection, our work is consistent with the previous recommendations by Mok et al 54 Additionally, we hypothesize that other effector domains, such as DddA homologs or chimeric deaminases, 16,[32][33][34][35][36] will also work well with unconstrained TALEs.…”

“…[16][17][18][19][20] In light of their rapid and accessible engineering, transcription activator-like effectors (TALEs) are the most commonly used DNA-binding domains in most current mitochondrial base editing technologies. 16,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] DddA-derived cytosine base editors (DdCBEs) consisting of pairs of mitochondrially targeted fusion proteins composed of a TALE, one half of a split dsDNA deaminase toxin (DddAtox), and a uracil glycosylase inhibitor, represent the most extensively developed tools for mtDNA editing. 16,[30][31][32][33][34] Based on prior work on TALE-DNA interactions in native contexts and in gene targeting applications in the nuclear compartment, [23][24][25][26][27][37][38][39][40][41][42][43][44][45][46][47] the target flexibility of DdCBEs on mtDNA is presumed to be constrained by the requirement of a 5' thymine (5'-T) in their TALE bi...…”