Table 1.

Limitations of Current Methods to Alter mtDNA in Cells

Technique	Limitations & Considerations
Mitochondrial targeting
mitoTALENs, mitoZFNs	Can be complex protein engineering; Targeted degradation, not reparative; Heteroplasmy shifting; 5 kbp size limitation if AAV vector used
CRISPR/Cas9	Deficient or inefficient guide RNA import; Low NHEJ and HR rates
Adeno-associated virus (AAV) transduction	5 kbp DNA size limit; Mitochondria targeting with unknown consequences; Compensatory and not reparative; Non-integrating; Unclear how mtDNA fragment is maintained and expressed episomally
RNA import	Inefficient; Mechanism not understood; Compensatory and not reparative
Mitochondria transfer
Nuclear/spindle/pronuclear/polar body transfer	Efficiency, technical, and ethical considerations; Pre-existing mtDNAs only
Co-culture with isolated mitochondria	Sporadic, low frequency
Transmitochondrial cytoplasmic fusions (cybrids)	Unwanted miRNAs, lncRNAs, proteins, organelles
Cell-to-cell transfer	Specific cell types and conditions; Unclear how to control transfer process; Mechanism(s) not fully understood
Microinjection*	Low efficiency; Microinjection needle prone to clogging; Needle imparts mechanical stress and cell trauma
Mitocytoplast fusion*	Low efficiency; Interspecies/fusion impurities
Photothermal nanoblade technology*	Operator and technique dependent
Other
Bottleneck to shift mtDNA heteroplasmy	Unclear if works with all cell types and mtDNA mutations; Stochastic at reprogramming rates

^*Potential approach to transfer mitochondria with modified mtDNA sequences