Mitochondrial genome engineering coming-of-age

Abstract

The mitochondrial genome has been difficult to manipulate because it is shielded by the organelle double membranes, preventing efficient nucleic acid entry. Moreover, mitochondrial DNA (mtDNA) recombination is not a robust system in most species. This limitation has forced investigators to rely on naturally occurring alterations to study both mitochondrial function and pathobiology. Because most pathogenic mtDNA mutations are heteroplasmic, the development of specific nucleases has allowed us to selectively eliminate mutant species. Several 'protein only' gene-editing platforms have been successfully used for this purpose. More recently, a DNA double-strand cytidine deaminase has been identified and adapted to edit mtDNA. This enzyme was also used as a component to adapt a DNA single-strand deoxyadenosine deaminase to mtDNA editing. These are major advances in our ability to precisely alter the mtDNA in animal cells.

Keywords: TALEN; gene editing; genetic engineering; mitochondria.

FIGURE 1.. The use of mitochondrial genome editors for the treatment of mitochondrial genetic disorders.

(A) Within the same cell, the sequence of some mtDNA molecules, denoted as mutant (MT), diverge from the wild type (WT) ones, usually by a single nucleotide polymorphism (SNP). This concept is called heteroplasmy and can be quantified by determining the percentage of WT versus MT alleles. (B) One approach to increase the amount of WT mtDNA molecules consists of introducing a MT-specific nuclease capable of linearizing MT mtDNA molecules. These linearized fragments will be subsequently degraded and removed from the pool of mtDNA molecules available for replication. (C) An alternative approach directly reverts the mutation of interest to generate WT mtDNA molecules from MT mtDNA molecules with the use of an appropriate mitochondrial-targeted DNA base editor.

KEY FIGURE 2:. The architecture of the different mitochondrial genome editors.

All the mtDNA editing platforms have added to N-terminus of their constructs a Mitochondrial Targeting Signal (MTS). Once in the mitochondrial matrix, their DNA binding domains (depicted in different shades of blue or red) will bind to their corresponding target DNA region (shaded in the same color). When required, this phenomenon will catalyze the dimerization and thus activation of the catalytic domains exclusively at the target site. (A) homodimeric Restriction endonucleases. (B) Heterodimeric mtZFN. A Nuclear Export Signal (NES) was also introduced to suppress the nuclear mis-localization of the constructs. (C) TALE-based nucleases. mitoTALEN is heterodimeric whereas I-TevI is monomeric and only digest target sites that follow a 5′-CNNNG-3′ motif. (D) mitoARCUS in monomeric as the original homodimer was fused into one polypeptide chain. (E) The applicability of CRISPR to mtDNA editing is still controversial. Even though most of the CRISPR binding affinity is determined by the sequence of the gRNA spacer (colored in red), the PAM-Interacting Domain of the Cas protein (PID, also in red) will further expand the targeted sequence to include an extra motif known as Protospacer Adjacent Motif (PAM). This PAM is species-specific. (F) Heterodimeric DddE can only deaminate with high efficiency cytosines that are preceded by a thymine. The UGI is an inhibitor of the mitochondrial Base-Excision repair pathway (mtBER). (G) DddA_tox is required for the system to work with the highest efficiency possible. Any given A can be targeted within the central region of the spacer sequence.