Expanding DdCBE-mediated targeting scope to aC motif preference in rat

Mitochondrial genome editing: strategies, challenges, and applications

“…In the cultured cell lines, utilizing various delivery strategies as previously mentioned, mtDNA base editors can target virtually all genes within the mtDNA, encompassing human, mouse, and rat cells. Furthermore, in vivo mtDNA base editing has been confirmed in various organisms, including mouse ( 46 , 48 , 50 , 55 , 61 , 62 , 70 ), zebrafish ( 63 , 64 ), rat ( 65 - 67 ), and even plant ( 72 ). However, the effectiveness of target achievement and efficiency may vary depending on the design rules of individual mtDNA base editors.…”

“…Electroporation is commonly used for transfecting various cell types, including human, mouse, rat, and patient-derived cells. In addition, the delivery of genome engineering tools in the form of mRNA into embryonic cells is a widely practiced method ( 24 , 46 , 48 , 55 , 61 - 67 ). This allows for mitochondrial genome editing in the early stages of embryonic development, and the generation of new models for genetic disease.…”

“…This allows for mitochondrial genome editing in the early stages of embryonic development, and the generation of new models for genetic disease. Through microinjection of embryos, mtDNA-associated disease models have been created using mitochondrial genome editing in mouse ( 46 , 48 , 55 , 61 , 62 ), zebrafish ( 63 , 64 ), and rat ( 65 - 67 ). Moreover, the efficiency and accuracy of mitochondrial genome editing have been evaluated by applying the microinjection method to clinically discarded human embryos with three pronuclei (3PN) or two pronuclei (2PN) ( 56 , 68 ).…”

Harnessing accurate mitochondrial DNA base editing mediated by DdCBEs in a predictable manner

“…As an alternative, TALE-based DdCBE is an all-protein base editor, which could immediately catalyze the deamination of cytidines within double-strand DNA. It has been reported that DdCBEs strongly prefer 5′-tC targets or even 5′-aC targets for mtDNA editing ( Boyne et al, 2022 ; Qi et al, 2023 ; Silva-Pinheiro et al, 2023 ), although some labs have expanded the scope of the target by constructing some other DddA variants ( Mok et al, 2022 ; Guo et al, 2023 ; Mi et al, 2023 ; Wei et al, 2023 ). Meanwhile, based on DdCBE, a series of related variants have been developed one after another, including DddAtox mutant-containing DdCBE with higher editing efficiency and broader editing scope ( Mok et al, 2022 ), transcription-activator-like effector-linked deaminases (TALEDs) which can introduce A-G substitution ( Cho et al, 2022 ) and strand-selective base editors (mitoBEs) with high A-G editing efficiency and specificity ( Yi et al, 2023 ), as well as strand-preferred base editor (CyDENT) with high strand-specificity and broader editing scope ( Hu et al, 2023 ).…”

Mitochondrial genome editing: Get aCcess to modeling broad disease mutations with engineered base editors

“…In this issue of Molecular Therapy – Nucleic Acids , Qi et al. 4 engineered canonical DdCBE to edit mtDNA within a broad sequence context. The authors achieved successful modification of several 5′-aC-3′ sequences and generated mitochondrial gene-edited rats with human-like symptoms.…”