Extended Data Fig. 1: Design of the MitoKO library.

a. Schematic representation of possible DdCBE orientations of the DddA_tox G1333 split and corresponding preference for base editing of cytosines within the DNA targeting window. The N and C-terminal fragments of the DddA_tox G1333 split can be linked to the TALE arrays binding either L-strand or H-strand of the mtDNA. The split orientation influences which cytosines are edited. b. The TALE designs to target all mouse protein-coding genes in the mouse mitochondrial genome. For each mtDNA protein-encoding gene, two TALEs binding the L-strand (L1 and L2) and two TALEs binding the H-strand (H1 and H2) of the mtDNA were designed to yield variable spacing windows, so that the target cytosine will be positioned at the center to reflect the preference of the DddA_tox G1333 split. c. Schematic of the general workflow for the screening of the MitoKO library involving transient transfection of cultured mouse NIH/3T3 cells with plasmids co-expressing DdCBE monomers and fluorescent marker proteins, followed by FACS-based selection of cells expressing both monomers and evaluation of mtDNA from DdCBE-treated cells, 7 days post-transfection.