DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome

Abstract

Critical mutations of mitochondrial DNA (mtDNA) generally lead to maternally inheritable diseases that affect multiple organs and systems; however, it was difficult to alter mtDNA in mammalian cells to intervene in or cure mitochondrial disorders. Recently, the discovery of DddA-derived cytosine base editor (DdCBE) enabled the precise manipulation of mtDNA. To test its feasibility for in vivo use, we selected several sites in mouse mtDNA as DdCBE targets to resemble the human pathogenic mtDNA G-to-A mutations. The efficiency of DdCBE-mediated mtDNA editing was first screened in mouse Neuro-2A cells and DdCBE pairs with the best performance were chosen for in vivo targeting. Microinjection of the mRNAs of DdCBE halves in the mouse zygotes or 2-cell embryo successfully generated edited founder mice with a base conversion rate ranging from 2.48% to 28.51%. When backcrossed with wild-type male mice, female founders were able to transmit the mutations to their offspring with different mutation loads. Off-target analyses demonstrated a high fidelity for DdCBE-mediated base editing in mouse mtDNA both in vitro and in vivo. Our study demonstrated that the DdCBE is feasible for generation of mtDNA mutation models to facilitate disease study and for potential treatment of mitochondrial disorders.

Keywords: DdCBE; base editing; mitochondrial disorder; mouse model; mtDNA.

Graphical abstract

Figure 1

DdCBE-mediated m.G7763A and m.G2820A mutations in mouse N2A cells (A and B) Sequence alignments indicate that mouse m.G7763 and m.G2820 are conserved with human m.G8363 and m.G3376, respectively. TALE targeting sequences are labeled in blue and the editing sites in red. (C–F) DdCBE-mediated editing efficiencies at m.G7763 (C and E) and m.G2820 (D and F) in mouse N2A cells. (C and D) Representative Sanger sequencing chromatograms of edited sites. (E and F) Deep sequencing analyses of editing efficiencies of different combinations of DdCBE pairs. Values and error bars reflect mean ± SEM of n = 3 independent biological replicates.

Figure 2

DdCBE-mediated m.G7763A and m.G2820A mutations in vivo (A and B) Representative Sanger sequencing chromatograms of low (upper) and high (lower) base conversion rate in m.G7763A (A) and m.G2820A (B) founder mice. (C–D) Base conversion rate of edited mice obtained through microinjections of DdCBE targeting m.G7763 (C, n = 25) and m.G2820 (D, n = 27). (E) Deep sequencing analyses of the base conversion rates in different tissues of m.G7763A edited founder mice 3# (blue) and 12# (red). (F) Representative Sanger sequencing chromatograms of low (upper) and high (lower) mutation loads in oocytes of m.G7763A edited founder mice 3# (left) and 12# (right).

Figure 3

Germline transmission of m.G7763A and m.G2820A mutations (A and B) Representative Sanger sequencing chromatograms of m.G7763A (A) and m.G2820A (B) founder mice and their offspring. (C and D) Deep sequencing analyses of the base conversion rate and mutation loads in m.G7763A (C) and m.G2820A (D) founder mice and their offspring. Results of the founder and its F1 offspring are shown in the same box. Red dots: founder; black dots: F1 offspring.

Figure 4

Off-target editing by DdCBE in vivo (A–D) Deep sequencing analyses of off-target editing rates in m.G7763A (A), m.G2820A (B), m.G12918A (C), and m.G7741A (D) founder mice (red dots) with wild-type mice as control (blue dots). Each dotted box indicates a single off-target site.

Figure 5

Off-target editing by DdCBE on entire mitochondrial genome. Whole mtDNA sequencing of m.G7763A and m.G2820A founder mice.