Fig. 1. MitoKO DdCBE library design strategy.

a, The architecture of DdCBE monomers used to generate the mitoKO library targeting each protein-coding gene of the mouse mtDNA. The mtDNA specificity is provided by programmable TALE domains. In each experiment, the DddA_tox G1333 split (purple) was tested in both orientations to achieve editing of the desired ‘TC’ sites. The MTS were from human superoxide dismutase 2 (SOD2) or cytochrome C oxidase subunit 8 (COX8). UGI, uracil glycosylase inhibitor. L-strand or (L), light mtDNA strand. H-strand or (H), heavy mtDNA strand. b, The strategy employed to ablate the 12 out of 13 mtDNA protein-encoded genes (Nd1, Nd2, Nd3, Nd4, Nd5, Nd6, Cytb, CoI, CoII, CoIII, Atp6 and Atp8) by introducing a premature stop codon with base editing. In the vertebrate mitochondrial genetic code, the TGA codon encodes tryptophan (Trp). Transition of the cytosine (C) in the opposite strand to a thymine (T) (in purple) using base editing leads to a premature TAA stop codon. c, The strategy employed to knock out the mtDNA protein-encoded gene Nd4l, by introducing a premature stop codon with base editing. Transition of the cytosine (C) in a CAA codon encoding glutamine (Gln) to a thymine (T) (in purple) using base editing leads to a premature TAA stop codon, thereby silencing the Nd4l gene. In this site, base editing can potentially edit the adjacent C of a GTC codon encoding valine (Val). However, the resulting GTT codon also encodes valine leading to a silent mutation.