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.

Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors

“…DdCBEs 1 and zinc finger deaminases (ZFDs) 2 -composed of the split interbacterial toxin DddA tox , a uracil glycosylase inhibitor (UGI), and custom-designed DNA-binding TALE arrays and zinc finger proteins, respectively-are highly versatile genome editing tools that enable targeted C-to-T conversions in the nuclear genome and mitochondrial [2][3][4][5][6][7][8][9] and chloroplast DNA [10][11][12] . DddA tox , an enzymatic moiety in the interbacterial toxin DddA derived from Burkholderia cenocepacia, catalyzes cytosine deamination within double-strand DNA (dsDNA) 13 .…”

DNA base editing in nuclear and organellar genomes

“…Applying this system to animal mitochondria, it was possible to create mice with base-edited mitochondrial genomes that persisted in a heterochondriomic state and were transmitted through the germline [166,167].…”

Modulating Mitochondrial DNA Heteroplasmy with Mitochondrially Targeted Endonucleases

“…These studies prove that the DdCBE platform now allows generation of transgenic animal models for mtDNA disease which have previously been lacking. 26,36 The methodology is ground breaking and has raised high hopes, however, a drawback to the use of the system may arise from unspecificity and off-target effects, as it was soon reported that the mt-DdCBEs lead to off-target effects both in the mitochondrial and nuclear genomes. Low frequency off-target changes in mtDNA seem to be fairly common but even more worrying is the substantial amount of off-target singlenucleotide changes seen in the nuclear genome, 73 which may limit the usability of the mt-DdCBEs at least for therapeutic purposes.…”