Various
mitochondrial diseases, including
mitochondrial encephalopathy,
lactic acidosis, and
stroke-like episodes (
MELAS), are associated with heteroplasmic mutations in mitochondrial
DNA (
mtDNA). Herein, we refined a previously generated G13513A
mtDNA-targeted
platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate
mtDNA heteroplasmy in
MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying
codons in their expression vectors could suppress the reduction in the
mtDNA copy number, which contributed to the rapid recovery of
mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover,
MELAS-iPSCs with a high proportion of G13513A mutant
mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally,
drug-inducible, myogenic differentiation 1 (MYOD)-transfected
MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into
myosin heavy chain-positive myocytes, with or without mutant
mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.