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

Abstract

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)—composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor—enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities

“…In contrast, analyses of nuclear off-target editing by enhanced DdCBEs [ 39 ], mDdCBEs [ 71 ], and mitoZFDs [ 58 ] have remained limited to targeted amplicon sequencing of a few predicted off-target sites. More fully assessing the levels and significance of these off-target edits is an important next step in the field, both in mtDNA and nDNA [ 39 , 57 , 58 , 63 , 71 , 72 ].…”

“…Excessive imprecision of mtDNA base editing technologies has the potential to limit their use for disease modeling and therapeutic applications. Therefore, aiming to circumvent this constraint, Kim lab [ 72 ] developed high-fidelity DdCBEs (HiFi-DdCBEs), which relied on interface-engineered split DddA tox variants. Wild-type split DddA tox halves can spontaneously reassemble independently of TALE-DNA interactions, leading to unwanted off-target mutations in nDNA or mtDNA [ 57 , 72 ].…”

“…Therefore, aiming to circumvent this constraint, Kim lab [ 72 ] developed high-fidelity DdCBEs (HiFi-DdCBEs), which relied on interface-engineered split DddA tox variants. Wild-type split DddA tox halves can spontaneously reassemble independently of TALE-DNA interactions, leading to unwanted off-target mutations in nDNA or mtDNA [ 57 , 72 ]. To address this issue, amino acid residues within the interface between split DddA tox halves were substituted with alanine, which has a chemically inert and non-bulky side chain [ 72 ].…”

“…Wild-type split DddA tox halves can spontaneously reassemble independently of TALE-DNA interactions, leading to unwanted off-target mutations in nDNA or mtDNA [ 57 , 72 ]. To address this issue, amino acid residues within the interface between split DddA tox halves were substituted with alanine, which has a chemically inert and non-bulky side chain [ 72 ]. These alanine substitutions resulted in split DddA tox variants that required DNA binding of their respective TALE arrays in order to reassemble and form a functional deaminase domain [ 72 ].…”

Chemical and Biological Approaches to Interrogate off-Target Effects of Genome Editing Tools

“…Lei et al demonstrated that fusing a nuclear export signal sequence (NES) can significantly reduce the genome-wide nuclear off-targets, and coexpression with a DddA inhibitor fused with nuclear localization signals (NLS) can eliminate both nuclear and mitochondrial off-targets with only a minor reduction in on-target efficiency . Lee et al created HiFi-DdCBEs by modifying amino acids around the binding interface between the two DddA halves, limiting functional deaminase activity to the intended target proximity and reducing collateral off-target changes …”