Research Project
9406532
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 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.
