I would also like to thank all other co-authors Roshan, @ChaligneRonan, Ignas, Rahul, Meril, Daphne, and @MarcoTigano for your generous help! Last but not least, none of this will be possible without @AgnelSfeir support and guidance along the way. 19/19.

This work is a collaborative effort between our lab (@TKavlashvili @edebitet Toby), and @themaxland from @dana_peer lab and SAIL team, Ruobing and Keun from Craig Thompson lab, and @minsoo_kim1 from @EdReznik lab, whom I enjoyed working with and have learned so much from! 18/19.

Although more work is needed to improve the precision and efficiency, we hope that our method will be useful to the community to study mtDNA deletions in various contexts. 17/19.

Finally, with help from @TKavlashvili and @edebitet, we demonstrated the flexibility of this method by engineering different types of mtDNA deletions (and even insertions) using site-specific nucleases and in iPSCs. 16/19.

Future work is necessary to understand the function and mechanism of these gene programs in heteroplasmic cells. 15/19.

For nuclear genes, we found two modes of gene expression alterations: one triggered by the heteroplasmy threshold, the other gradually responding to heteroplasmy. 14/19

For the mitochondria encoded genes, while most of the mRNAs decrease with increasing heteroplasmy, two mRNAs (ND1 and ND2) were enriched in high heteroplasmic cells. 13/19.

With @themaxland spearheading the analysis of single cell data, we made a few interesting observations on mitochondrial and nuclear gene expression. 12/19.

We next wanted to investigate the transcription programs responding to deletion heteroplasmy levels. In collaboration with the SAIL team at MSK, we took a multiplexing approach by pooling ~46 clones and profiling mtDNA genotypes and transcriptome in single cells. 11/19

Thanks to the help from Ruobing and Keun from Craig Thompson's lab, we showed that OXPHOS components and function, and TCA metabolites were drastically declined in clones with >=75% deletions, suggesting a threshold effect of the ScaI deletions in RPE cells. 10/19

Isolation of clonal cells further confirmed the heterogeneity. But more importantly, we obtained a panel of clones at a wide range of deletion heteroplasmy, allowing us to investigate how heteroplasmy levels affect cellular and mitochondrial functions. 9/19

At the time, Toby in our lab has repeatedly asked me: Do all the cells in the population have the same level of deletions? She showed me to use FISH to measure heteroplasmy at the single cell level. We quickly realized the heterogeneity of deletion levels across cells. 8/19

Although we were able to induce deletion formation, the levels we could get were far from the levels (>60%) in mitochondrial diseases. 7/19.

We are not the first to come up with the idea. This approach has been used elegantly in Drosophila to study mtDNA deletions in the flight muscle 6/19.

This inspired us to leverage the approach of “mito-DSB + mito-End-joining” to create mammalian cell models for mtDNA deletions, a major cause of mitochondrial diseases. 5/19

Because we used a multi-cutter mito-ScaI, the small cleaved fragments were often lost, leading to formation of large deletions. 4/19

We started by testing simple end joining systems from bacteria and phage. With a restriction enzyme to cleave mtDNA, we found that two end joining systems – Ku and LigD from Mycobacteria and T4 DNA ligase from T4 bacteriophage – could ligate broken DNA in the mitochondria! 3/19.

The project started when we were thinking about how mitochondria deal with mtDNA breaks. However, mammalian mitochondria seem to lack active DSB repair and tend to degrade broken mtDNA. We were curious what happens if we provide mitochondria with an DSB repair machinery. 2/19.

Excited to share my PhD work in @Agnelsfeir’s lab describing a method to engineer mitochondrial DNA deletions in human cells and our exploration of how cellular metabolism and transcription respond to deletion heteroplasmy. 1/19.

RT @zhtianp2: I’m thrilled to share that I will be joining Department of Radiation Oncoloy (and BMG) @UVA as AP this August. The Zhang Lab….