Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing

DdCBE-mediated mitochondrial base editing in human 3PN embryos

“…These mutations lead to human mitochondrial disorders such as LHON, MERRF, Leigh Syndrome, and MELAS (Supplementary Table S1 ). For each site, 4 pairs of DdCBE plasmids with different combinations of orientation and split type (Left-G1333C (L1333C) + Right-G1333N (R1333N), L1397C + R1397N, L1333N + R1333C, L1397N + R1397C) were constructed using the DdCBE assembly kit we developed recently (Supplementary Table S2 and Data S1 ) 8 , 9 . HEK293FT cells were transfected with each DdCBE pair and collected on day 6 to amplify the targeted region of mtDNA for Sanger sequencing.…”

Human cleaving embryos enable efficient mitochondrial base-editing with DdCBE

“…Recently, DddA-derived cytosine base editors (DdCBEs) have been developed to specifically induce C-to-T conversion in mtDNA by fusion of sequence-programmable TALE and split deaminase derived from interbacterial toxins 10 . DdCBE has been demonstrated to generate mutant zebrafish and mice carrying base-modified mtDNA by injecting DdCBE mRNA into zygotes 11 , 12 . However, the efficacy of DdCBE for specific installation of C-to-T conversion in mtDNA of human embryos remained to be investigated.…”

DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome

“…DdCBE vectors were assembled by one-step Golden Gate assembly using an RVD library containing 192 vectors we developed before. 27 Transfection of DdCBE halves led to successful G-to-A conversion at both loci ( Figures 1 C and 1D). Deep sequencing data indicated that the combination of left-1397C with right-1397N achieved the highest editing efficiency for both m.G7763A (23.07% ± 4.54%) and m.G2820A (35.63% ± 2.18%) targeting ( Figures 1 E and 1F).…”

“…DdCBE vectors were assembled using RVD libraries as before. 27 The complete set of plasmids for assembling DdCBE can be obtained from Addgene (pending). In brief, RVDs and backbone plasmids were digested with Bsa I and ligated with T4 DNA ligase in a single reaction using the following program: 37°C for 10 min; 10 cycles of 10 min at 37°C and 10 min at 16°C; 50°C for 5 min; 80°C for 5 min.…”