sfeir, a. | Search bioRxiv

bioRxiv2025

RNA Transcripts Serve as a Template for Double-Strand Break Repair in Human Cells

Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Pol{zeta}) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

molecular biology↗

bioRxiv2024

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a [~]3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of [~]75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/618543v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@ab900corg.highwire.dtl.DTLVardef@17e094dorg.highwire.dtl.DTLVardef@194a10corg.highwire.dtl.DTLVardef@d925e5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICombining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cells C_LIO_LIEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy C_LIO_LI75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunction C_LIO_LITwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing C_LI

molecular biology↗