Effective engineering of the mitochondrial genome

Abstract 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 kb deletions spanning position to position 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 protein encoding components of the mitochondrial genome and each of the encoded ribosomal RNAs rRNA in both mouse and human cells 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