Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized

Engineering of mammalian mtDNA has been hampered by an inability to import nucleic acids into mitochondria. A limited toolkit exists for manipulation of mammalian mtDNA, relying on protein-only nucleolysis and heteroplasmy-shifting approaches. Although present in lower metazoans, the weight of evidence against an efficient endogenous RNA import mechanism in mammalian mitochondria is considerable. Controversially, the application of CRISPR/Cas9 for manipulation of mammalian mtDNA in human cells has been reported. In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial. In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial. Biological understanding of complex organisms in the modern era relies heavily on reverse genetics. As an area of interest for many, a robust method for directed genetic manipulation of mammalian mitochondria has been sought for several decades. More recently, efforts to this end have largely focused on the search for treatments of mitochondrial disease. Incurable and largely intractable, mitochondrial diseases caused by mutation of the mitochondrial genome affect approximately one in 5000 and represent a substantial disease burden [1Gorman G.S. et al.Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.Ann. Neurol. 2015; 77: 753-759Crossref PubMed Scopus (529) Google Scholar]. The dawn of the genome-editing era augurs well for both basic and clinical mitochondrial research, and the CRISPR/Cas9 revolution in particular seems to bring a paradigm shift within our grasp. However, fundamental questions regarding the capacity of mammalian mitochondria to import the guide RNA (gRNA; see Glossary) molecules needed for a viable CRISPR/Cas9 system cast doubt upon such an enterprise. Over recent years evidence against the notion of endogenous import of nucleus-encoded RNA into mammalian mitochondria has accrued. In this article we discuss the mitochondrial genetic system, evidence for and against endogenous RNA import into, and proposed functions within, mammalian mitochondria, and recent efforts towards genetic manipulation of mitochondria, including the controversial report of a mitochondrial CRISPR/Cas9 system. From the initial alphaproteobacterial engulfment, that formed the first eukaryote through endosymbiosis, to the present-day organelle residing in mammalian cells, the relationship between mitochondria and their hosts has evolved substantially. Where once mitochondria-like symbionts were advantageous principally for their capacity to harness redox chemistries, the role of mitochondria in diverged eukaryotes, such as mammals, is much more intricately embedded in essential organismal function. Facilitation of these functions relies upon an electrochemical disequilibrium potential across the inner mitochondrial membrane (IMM) that is generated through proton pumping by respiratory chain complexes I, III, and IV. Taken together, the respiratory chain and ATP synthase consist of ∼90 protein subunits, forming IMM-bound protein complexes. The vast majority of these proteins are encoded in and expressed from the nuclear genome; however, a subset is encoded within a spatially and heritably separate genome – the mitochondrial genome. Mammalian mitochondrial DNA (mtDNA) is a multi-copy, circular, double-stranded DNA molecule encoding 13 essential membrane-bound polypeptide subunits of the respiratory chain complexes I, III, IV, and ATP synthase, 22 tRNAs, and two ribosomal RNAs (rRNAs). At ∼16.5 kb, mammalian mtDNAs are relatively small and genetically compact, containing very little non-coding sequence and two overlapping genes [2Anderson S. et al.Sequence and organization of the human mitochondrial genome.Nature. 1981; 290: 457-465Crossref PubMed Scopus (7611) Google Scholar]. The mitochondrial genome is packaged into individual nucleoids that consist principally of the mitochondrial transcription factor A (TFAM) [3Kukat C. et al.Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.Proc. Natl. Acad. Sci. U. S. 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In placental mammals a full complement of 22 functional tRNA species capable of recognizing 60 sense codons, and two rRNAs that are required for translation by mitoribosomes, are also encoded in mtDNA. Considering the substantial structural differences between mitochondrial and cytosolic tRNAs, the divergence and incompatibility of codon usage between mitochondrial and nuclear mRNAs, the lack of unassigned codons in mitochondrial open reading frames (ORFs) [22Suzuki T. et al.Human mitochondrial tRNAs: biogenesis, function, structural aspects, and diseases.Annu. Rev. Genet. 2011; 45: 299-329Crossref PubMed Scopus (376) Google Scholar], and that all other mitochondrial proteins are encoded and expressed from the nuclear genome, any mRNA-decoding function for RNA imported into mitochondria is not immediately apparent. However, various other roles for endogenous, nuclear-encoded RNAs imported into mitochondria have been debated (Figure 1A,B). An unusual, bacteria-like feature of mammalian mitochondrial gene expression is the near-unit length polycistronic transcripts produced through transcription of mtDNA. Within the polycistrons, most gene products are punctuated by one or more tRNAs, which require endonucleolytic processing at both 5′ and 3′ ends to release individual transcripts, a concept termed ‘tRNA punctuation’ [23Ojala D. et al.tRNA punctuation model of RNA processing in human mitochondria.Nature. 1981; 290: 470-474Crossref PubMed Scopus (1851) Google Scholar]. Essential to this process is mitochondrial RNase P (mtRNase P). Both nuclear and mitochondrial RNases P liberate the 5′ ends of immature tRNA transcripts through structure-guided endonucleolytic processing. RNase P is an ancient enzyme, initially identified in bacteria, followed by eukaryotic nuclei and yeast mitochondria [24Robertson H.D. et al.Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic acid presursor.J. Biol. Chem. 1972; 247: 5243-5251PubMed Google Scholar, 25Koski R.A. et al.Identification of a ribonuclease P-like activity from human KB cells.Cell. 1976; 9: 101-116Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 26Hollingsworth M.J. Martin N.C. RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.Mol. Cell. Biol. 1986; 6: 1058-1064Crossref PubMed Scopus (76) Google Scholar]. 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Human mtRNase P consists of three protein subunits: a mitochondrially targeted tRNA m1R methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehydrogenase/reductase (SDR) family, SDR5C1 (HSD17B10, MRPP2), and a protein with homology to PiIT N terminus (PIN) domain-like metallonucleases, PRORP (MRPP3) [31Holzmann J. et al.RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme.Cell. 2008; 135: 462-474Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 32Vilardo E. et al.A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase – extensive moonlighting in mitochondrial tRNA biogenesis.Nucleic Acids Res. 2012; 40: 11583-11593Crossref PubMed Scopus (150) Google Scholar]. Importantly, the human mtRNase P was shown specifically not to contain any trans-acting RNA. This paradigm-shifting subclass of proteinaceous RNase P (PRORP) has since been identified in most eukaryal lineages ([33Rossmanith W. Of P and Z: mitochondrial tRNA processing enzymes.Biochim. Biophys. Acta. 2012; 1819: 1017-1026Crossref PubMed Scopus (78) Google Scholar, 34Lechner M. et al.Distribution of ribonucleoprotein and protein-only RNase P in Eukarya.Mol. Biol. Evol. 2015; 32: 3186-3193PubMed Google Scholar] for an in-depth review on the discovery and evolution of PRORPs). A further controversy concerning mitochondrial RNA processing by imported endogenous RNAs concerns the nRNase P-related endonuclease, the mitochondrial RNA processing ribonuclease (RNase MRP). Similarly to nRNase P, RNase MRP possesses a RNA subunit (termed 7-2 RNA in the early literature) and several protein components, most of which are shared with nRNase P [35Rosenblad M.A. et al.Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes.Nucleic Acids Res. 2006; 34: 5145-5156Crossref PubMed Scopus (59) Google Scholar]. RNase MRP was first described as a ribonucleoprotein complex present in mitochondria that is involved in the formation of a RNA primer during initiation of mammalian mtDNA replication [36Chang D.D. Clayton D.A. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA.Science. 1987; 235: 1178-1184Crossref PubMed Scopus (221) Google Scholar]. 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In addition, in vitro reconstitution experiments have suggested an RNase MRP-independent mechanism for primer processing in mtDNA replication, where the 3'-end of the RNA primer is generated by site-specific termination of transcription owing to G-quadruplex formation in nascent RNA, rather than cleavage by RNase MRP [40Wanrooij P.H. et al.G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 16072-16077Crossref PubMed Scopus (121) Google Scholar]. These findings point away from the requirement for non-mtDNA transcribed RNA to be present in mitochondria for RNA processing, suggesting that endogenous RNA import into mammalian mitochondria is not required for normal cellular functions. Another area of debate concerning mitochondrial import of endogenous RNA in mammals focuses on the RNA content of mitoribosomes, specifically the existence of a minor structural rRNA species analogous to the 5S rRNA that is found in ribosomes from other cellular compartments and organisms. Several groups have argued in favor of 5S rRNA being present in mammalian mitochondria [41Yoshionari S. et al.Existence of nuclear-encoded 5S-rRNA in bovine mitochondria.FEBS Lett. 1994; 338: 137-142Crossref PubMed Scopus (56) Google Scholar, 42Magalhaes P.J. et al.Evidence for the presence of 5S rRNA in mammalian mitochondria.Mol. Biol. Cell. 1998; 9: 2375-2382Crossref PubMed Scopus (102) Google Scholar, 43Entelis N.S. et al.5S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements.J. Biol. 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PNPase is a homotrimeric 3′–5′ exoribonuclease which, together with mitochondrial RNA-specific helicase, hSUV3, forms the RNA degradasome in the mitochondrial matrix [54Borowski L.S. et al.Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci.Nucleic Acids Res. 2013; 41: 1223-1240Crossref PubMed Scopus (132) Google Scholar, 55Chujo T. et al.LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and promotes polyadenylation in human mitochondria.Nucleic Acids Res. 2012; 40: 8033-8047Crossref PubMed Scopus (113) Google Scholar]. However, an alternative function and localization of PNPase has been proposed. Detection of PNPase in the mitochondrial intermembrane space (IMS), rather than in the matrix, has led to suggestions that it could mediate mitochondrial matrix translocation of 5S rRNA, H1 RNA, 7-2 RNA, and more recently also microRNAs (miRNAs) by an uncharacterized mechanism [56Wang G. et al.PNPASE regulates RNA import into mitochondria.Cell. 2010; 142: 456-467Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 57Shepherd D.L. et al.Exploring the mitochondrial microRNA import pathway through polynucleotide phosphorylase (PNPase).J. Mol. Cell. Cardiol. 2017; 110: 15-25Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar]. This was surprising because PNPases, an ancient family of enzymes, had previously been found to reside in the matrix and to be involved in degradation of RNA, rather than in transport [54Borowski L.S. et al.Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci.Nucleic Acids Res. 2013; 41: 1223-1240Crossref PubMed Scopus (132) Google Scholar, 58Piwowarski J. et al.Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria.J. Mol. Biol. 2003; 329: 853-857Crossref PubMed Scopus (71) Google Scholar, 59Antonicka H. Shoubridge E.A. Mitochondrial RNA granules are centers for posttranscriptional RNA processing and ribosome biogenesis.Cell Rep. 2015; 10: 920-932Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 60Rhee H.W. et al.Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (727) Google Scholar]. 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Given the consensus localization of PNPase in the mitochondrial matrix [100Rhee H.W. et al.Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (260) Google Scholar], its well-described role in mitochondrial RNA degradation, the lack of a well-understood RNA import mechanism, and the likely dispensable role of RNAs it is alleged to transport, PNPase-mediated RNA import into mammalian mitochondria is not widely accepted, and requires further exploration and confirmation. In addition to the research concerning import of endogenous RNAs into mammalian mitochondria, discussed above, there also exists a less well interrogated literature suggesting both import and export of miRNAs [62Zhang X. et al.MicroRNA directly enhances mitochondrial translation during muscle differentiation.Cell. 2014; 158: 607-619Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar], long non-coding RNAs (lncRNAs) [63Dietrich A. et al.Organellar non-coding RNAs: emerging regulation mechanisms.Biochimie. 2015; 117: 48-62Crossref PubMed Scopus (45) Google Scholar], and tRNAs [64Rubio M.A. et al.Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9186-9191Crossref PubMed Scopus (115) Google Scholar] into and from mammalian mitochondria, which will not be discussed here because we believe this requires validation by independent studies. It is helpful to underscore the often-contradictory findings reported in the studies discussed above by reference to valuable data from a comprehensive, quantitative analysis of the human mitochondrial transcriptome [65Mercer T.R. et al.The human mitochondrial transcriptome.Cell. 2011; 146: 645-658Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar]. In this study, numerous nucleus-encoded RNA species were detected in enriched mitochondrial RNA samples, although upon disruption and removal of the outer mitochondrial membrane these RNAs were almost exclusively found to be less abundant or only fractionally enriched; by contrast, bona fide mtDNA-encoded RNA species were enriched by many orders of magnitude. Such outer-membrane contaminants have been long discussed [66Lightowlers R.N. et al.Mitochondrial protein synthesis: figuring the fundamentals, complexities and complications, of mammalian mitochondrial translation.FEBS Lett. 2014; 588: 2496-2503Crossref PubMed Scopus (48) Google Scholar], and are a likely source of confusion and controversy within the field. Despite innumerable innovative investigations that have yielded a substantial but mosaic literature on the subject, the vast majority of approaches to mitochondrial genome engineering have failed to be either efficient or robust, and progress has been glacial (as recently reviewed [67Patananan A.N. et al.Modifying the mitochondrial genome.Cell Metab. 2016; 23: 785-796Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]). Presently, reliable methods for the transformation of mitochondria exist only for yeasts [68Fox T.D. et al.Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA.Proc. Natl. 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