This proposal leverages our novel, unprecedented, and unique method for editing the mitochondrial genome. B-MoGen has exclusive license to this technology. This proposal will finally provide the final piece to complete the human genome project from a functional perspective since it has been impossible to make targeted gene knockouts in the mitochondrial genome until now. This is critically important given that the mitochondria play pivotal roles in human biology and disease. Indeed, there are many mitochondrial genetic diseases that have been impossible to model in isogenic pairs of cells. In this proposal, we will generate human cell lines, including a human induced pluripotent stem (iPS) cell line, that carry a mitochondrial genome deletion present in the most common human mitochondrial genetic disease called Kearns-Sayre Syndrome (KSS). We will employ our mitoTALE-nickases to introduce 4.9kb deletions spanning position 8469 to position 13147 in the mitochondrial genomes in these cells. We will employ our mitoTALENs to induce heteroplasmic shift of the mitochondrial genomes to those carrying the deletion. Secondly, we will develop tools to make doxycycline and cumate inducible mitoTALEN vectors for allowing conditional generation of mitochondrial deletions and induction of heteroplasmic shift. Finally, we will generate mitoTALE-nickases to target each of the 13 protein encoding components of the mitochondrial genome and each of the 2 encoded ribosomal RNAs (rRNA) in both mouse and human cells.
Public Health Relevance Statement: Project Narrative Although there have been significant advancements in gene editing over the past two decades, none have been able to precisely edit the mitochondrial genome, until now. The goal of the proposed project is to use mitochondrial gene editing technology we have acquired to generate a set of tools and reagents to create human and mouse cell lines that carry a deletion in each protein and ribosomal RNA encoded on the mitochondrial genome. The result of this proposal will provide researchers with a novel set of tools necessary to better understand and interrogate mitochondria biology and disease.
Project Terms: Animal Model; Animals; Autoimmunity; base; Biological; Biological Assay; Biology; Cardiovascular Diseases; Cell Line; Cell physiology; Cells; Clinic; commercial application; CRISPR library; Data; design; Development; Disease; disease-causing mutation; DNA; DNA Binding; DNA Double Strand Break; Doctor of Philosophy; Doxycycline; endonuclease; Endonuclease I; Engineering; experience; functional genomics; Gene Deletion; gene function; Gene Targeting; Generations; Genes; Genetic; Genetic Models; Genome; genome editing; Genome engineering; genomic tools; Genomics; Goals; Guide RNA; Hereditary Disease; homologous recombination; Human; Human Biology; Human Cell Line; human disease; Human Genome Project; induced pluripotent stem cell; inducible gene expression; insertion/deletion mutation; Kearns-Sayre syndrome; knockout gene; Knockout Mice; L Cells; Lentivirus Vector; Licensing; Mammalian Cell; Methods; Mitochondria; Mitochondrial DNA; mitochondrial genome; Modeling; Mouse Cell Line; Mus; Mutation; Neoplasm Metastasis; novel; novel strategies; Nuclear; nuclease; Patients; Phase; phase 2 study; Play; Positioning Attribute; Process; Proteins; Reagent; Reproducibility; Research; Research Personnel; Resources; Ribosomal Proteins; Ribosomal RNA; Role; Sales; Services; Site; T-Lymphocyte; Technology; Testing; tool; tool development; transcription activator-like effector nucleases; vector; Vertebrates; whole genome; Work
