Manipulating and elucidating mitochondrial gene expression with engineered proteins

Abstract

Many conventional, modern genome engineering tools cannot be used to study mitochondrial genetics due to the unusual structure and physiology of the mitochondrial genome. Here, we review a number of newly developed, synthetic biology-based approaches for altering levels of mutant mammalian mitochondrial DNA and mitochondrial RNAs, including transcription activator-like effector nucleases, zinc finger nucleases and engineered RNA-binding proteins. These approaches allow researchers to manipulate and visualize mitochondrial processes and may provide future therapeutics. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.

Structural basis of mitochondrial translation

“…Taken together, these pathomechanisms involve different stages of the mitoribosome activities, which have not been thoroughly investigated. Although advanced approaches to visualize mitochondrial processes have been developed ( Wallis et al, 2020 ), the basic molecular mechanism of how human mitoribosome orchestrates the flow of genetic information from mt-DNA encoded genes to functional proteins is yet to be characterized. Since recent work demonstrates that employment of cell signalling molecules and networks has a potential of targeted therapies for mitochondrial diseases ( Ferreira et al, 2019 ), the basic understanding of the underlying molecular mechanisms is crucial.…”

Mechanisms and Strategies for Determining m6A RNA Modification Sites by Natural and Engineered m6A Effector Proteins

“…However, the advantages of using PUFs include the small size of the PUF expression vector compared to genes of large Cas‐fused proteins that are difficult to pack into adeno‐associated virus vectors and the simplicity of the system without additional guide RNA. In addition, although CRISPR‐Cas‐based systems cannot be applied in mitochondria because of the difficulty of importing exogenous guide RNAs into mitochondria, [81] PUF‐based RNA manipulation can be achieved in mitochondria [80,82,83] …”

“…To achieve selectivity for specific endogenous mRNAs, it is desirable to use modified PUFs that can recognize longer sequences [64,67–69] . Notably, PUFs can target RNAs in mitochondria, which cannot be achieved using CRISPR‐Cas [83] . Although the presence of m 6 A has been reported in plant mitochondrial transcripts, [88,89] its presence in mammalian cells has not been observed, [90] but PUF has the potential to control mitochondrial RNA modifications.…”

Linking the mitochondrial genotype to phenotype: a complex endeavour

“…Wallis et al [43] review new methods for studying the genotype-phenotype link by manipulating mitochondrial gene expression with engineered proteins. Many genome engineering tools used for nuclear genome modification cannot be used to study mitochondrial genetics owing to the unusual structure and physiology of the mitochondrial genome.…”

“…Although challenges in the manipulation of mitochondria persist, new approaches are developed to modify the levels of mutant mammalian mitochondrial DNA and mitochondrial RNAs. Wallis et al [43] review methodssuch as restriction enzymes targeted to mitochondria (mitoREs), zinc finger proteins fused with a nuclease targeted to mitochondria (mtZFNs), transcription activator-life effectors fused with a nuclease targeted to mitochondria (mitoTALENs) and RNA-binding proteins engineered to target specific mitochondrial RNA-that allow us to manipulate mtDNA, to modulate mitochondrial gene expression and to track and visualize mitochondrial processes, and whose application and study may provide highly specific and customizable genetic tools that could be applied in future therapeutics.…”