Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Hussain, Syed-Rehan A.; Yalvaç, Mehmet Emir; Khoo, Bendict; Eckardt, Sigrid; McLaughlin, K. John

doi:10.1101/2020.02.11.944819

Cited by 17 publications

(21 citation statements)

References 41 publications

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“…A recent preprint study reported the editing of the MT-ND4 gene accomplished by targeting the guide RNA to an RNA transport derived stem loop element (RP-loop), while expressing the Cas9 enzyme with a mitochondrial localization sequence [ 363 ]. However, due to the controversial nature of mammalian mitochondrial RNA import [ 364 ], the use of the CRISPR/Cas9 application for mtDNA editing is still debated.…”

Section: Precision Medicine Approaches For Pmd Caused By Mtdna Defmentioning

confidence: 99%

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

et al. 2020

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Primary mitochondrial diseases (PMD) refer to a group of severe, often inherited genetic conditions due to mutations in the mitochondrial genome or in the nuclear genes encoding for proteins involved in oxidative phosphorylation (OXPHOS). The mutations hamper the last step of aerobic metabolism, affecting the primary source of cellular ATP synthesis. Mitochondrial diseases are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. The limited information of the natural history, the limitations of currently available preclinical models, coupled with the large variability of phenotypical presentations of PMD patients, have strongly penalized the development of effective therapies. However, new therapeutic strategies have been emerging, often with promising preclinical and clinical results. Here we review the state of the art on experimental treatments for mitochondrial diseases, presenting “one-size-fits-all” approaches and precision medicine strategies. Finally, we propose novel perspective therapeutic plans, either based on preclinical studies or currently used for other genetic or metabolic diseases that could be transferred to PMD.

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Section: Precision Medicine Approaches For Pmd Caused By Mtdna Defmentioning

confidence: 99%

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

et al. 2020

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“…A recent study showed that gRNAs with short hairpin structures can promote mitochondrial import and specific cleavage of mitochondrial DNA, albeit at low levels because of limited import into the mitochondrial matrix ( Loutre et al., 2018 ). More recently, Hussain et al. (2020) demonstrated that gRNA could be efficiently imported to the mitochondria of mammalian cells with an additional 20-bp stem–loop element of nuclear RNase P in a polynucleotide phosphorylase-dependent manner ( Wang et al., 2010 ).…”

Section: Toward Molecular Breeding Through Mitochondrial Genome Modificationmentioning

confidence: 99%

Advancing organelle genome transformation and editing for crop improvement

Chang

Jiang

2021

Plant Communications

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Plant cells contain three organelles that harbor DNA: the nucleus, plastids, and mitochondria. Plastid transformation has emerged as an attractive platform for the generation of transgenic plants, also referred to as transplastomic plants. Plastid genomes have been genetically engineered to improve crop yield, nutritional quality, and resistance to abiotic and biotic stresses, as well as for recombinant protein production. Despite many promising proof-of-concept applications, transplastomic plants have not been commercialized to date. Sequence-specific nuclease technologies are widely used to precisely modify nuclear genomes, but these tools have not been applied to edit organelle genomes because the efficient homologous recombination system in plastids facilitates plastid genome editing. Unlike plastid transformation, successful genetic transformation of higher plant mitochondrial genome transformation was tested in several research group, but not successful to date. However, stepwise progress has been made in modifying mitochondrial genes and their transcripts, thus enabling the study of their functions. Here, we provide an overview of advances in organelle transformation and genome editing for crop improvement, and we discuss the bottlenecks and future development of these technologies.

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“…Because all three intronic sequences do not include any known miRNAs or predicted stem-loop structures, they seem to act in trans as long ncRNA regulatory elements [20]. Similarly, constructs containing common selection markers/reporter genes like GFP, EGFP, Neo r , LacZ, and DsRed are often left within the target genome postselection [9,21,29,37,62]. However, these genes themselves can become potential targets of miRNAs of host origin, e.g., Mus musculus as discussed later.…”

Section: The Problemmentioning

confidence: 99%

“…Neomycin resistance gene (Neo r ) is one of the widely utilized selection markers for the cells which are correctly targeted, and the neomycin cassette itself is normally left within the genome post-selection, assuming that it has no adverse effect on the eukaryotic cell biology [21,50,62]. But upon careful observation, it can be seen that the Neo r gene construct itself is a potential target of several miRNAs of the eukaryotic origin or more specifically the miRNAs within the cells of the neomycin cassette containing transgenic mice (Table 1).…”

Section: Commonly Used Foreign Genes Targeted By Mus Musculus Mirnasmentioning

confidence: 99%

Inadvertent nucleotide sequence alterations during mutagenesis: highlighting the vulnerabilities in mouse transgenic technology

Ghosh

Chakrabarti

Shukla

2021

Journal of Genetic Engineering and Biotechnology

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In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as “junk DNA,” have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.

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Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Cited by 17 publications

References 41 publications

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

Advancing organelle genome transformation and editing for crop improvement

Inadvertent nucleotide sequence alterations during mutagenesis: highlighting the vulnerabilities in mouse transgenic technology

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