Mitochondrial DNA (mtDNA) diseases are multi-systemic disorders caused by mutations affecting a fraction or the entirety of mtDNA copies. Currently, there are no approved therapies for the majority of mtDNA diseases. Challenges associated with engineering mtDNA have in fact hindered the study of mtDNA defects. Despite these difficulties, it has been possible to develop valuable cellular and animal models of mtDNA diseases. Here, we describe recent advances in base editing of mtDNA and the generation of three-dimensional organoids from patient-derived human-induced pluripotent stem cells (iPSCs). Together with already available modeling tools, the combination of these novel technologies could allow determining the impact of specific mtDNA mutations in distinct human cell types and might help uncover how mtDNA mutation load segregates during tissue organization. iPSC-derived organoids could also represent a platform for the identification of treatment strategies and for probing the in vitro effectiveness of mtDNA gene therapies. These studies have the potential to increase our mechanistic understanding of mtDNA diseases and may open the way to highly needed and personalized therapeutic interventions.

Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to β-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to "calm down" its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Life's gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.

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