Fig. 3: Mitochondrial stresses and DSBs exacerbate mtDNA integration into the nuclear genome.

a Illustration of mitochondrial stresses and DSBs-induced mt-nuclear DNA fusions captured by PEM-seq and Insert-seq. CRISPR-Cas9 (scissor) targets the c-MYC locus in nuclear DNA, and the primer (purple arrow) for PEM-seq is adjacent to the target site. b Mitochondrial stresses inducing mt-nuclear DNA fusions captured by PEM-seq. Top: cells were transfected with a plasmid containing Cas9 and gRNA targeting c-MYC for 8 hours to allow the assembly of Cas9 and gRNA. Subsequentially, cells are treated with CCCP or paraquat for 24 or 48 hours , respectively, and then harvested for PEM-seq and Insert-seq analysis. Bottom: percentages of mt-nuclear DNA fusions captured by PEM-seq. Each dot represents a biological replicate. Mean ± SD; two-sided t-test; n = 3. Source data are provided as a Source Data file. c Distribution of mt-nuclear DNA fusion junctions captured by the c-MYC bait (black triangle) with or without mitochondrial stresses (untreated, gray bars; CCCP, light purple bars; paraquat, dark purple bars) treatment. Legends of mtDNA annotations are described as depicted in Fig.1c. The inner circles show the number of each mtDNA-nuclear DNA fusion point on mtDNA in a log scale. MT, mtDNA. d Frequency of PEM-seq-captured mt-nuclear DNA fusion junctions with or without mitoTALEN treatment. Each dot represents a biological replicate. Mean ± SD; two-sided t-test; n = 3. Source data are provided as a Source Data file. e Distribution of mt-nuclear DNA fusion junctions captured by the c-MYC bait (black triangle) with or without mitoTALEN (ND4 site, red triangle) treatment. Legends of mtDNA annotations are described as depicted in Fig. 1b. f Workflow of Insert-seq to enrich insertions (orange lines) at the Cas9-editing site (c-MYC locus). Briefly, two rounds of targeted PCR (purple and red arrows) are used to clone the editing events around the target site, followed by two rounds of size selection that enriches insertions. Source data are provided as a Source Data file. g Percentages of mtDNA integrations within total insertions captured by Insert-seq at the c-MYC locus in the presence or absence of mitochondrial stresses. Each dot represents a biological replicate. Mean ± SD; two-sided t-test; n = 3. h Percentages of mtDNA integrations within total insertions captured by Insert-seq at the c-MYC site. Each dot represents a biological replicate. Mean ± SD; two-sided t-test; n = 3.