Biology subjects

Crossley, M. P.

Publications and source records attributed to Crossley, M. P..

5 recordsLinked to original sources

bioRxiv2026

Protocol for in vivo DNA-RNA hybrid immunoprecipitation sequencing and analysis from frozen mammalian tissues

DNA-RNA hybrids (R-loops) form transiently on the genome and regulate cellular homeostasis. They also influence genome editing outcomes, highlighting their therapeutic potential in vivo. This protocol enables high-resolution mapping of DNA-RNA hybrids directly from frozen mouse tissues. Following tissue homogenisation and lysis, genomic DNA is extracted, digested and DNA-RNA hybrids are isolated using the hybrid-specific S9.6 monoclonal antibody. The purified hybrids are then processed for whole-genome sequencing to generate R-loop profiles. For complete details on the use and execution of this protocol, please refer to Puzzo, Crossley et al1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/716701v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@3d8f86org.highwire.dtl.DTLVardef@199cd84org.highwire.dtl.DTLVardef@83c51eorg.highwire.dtl.DTLVardef@1024332_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOgraphical abstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3 end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

molecular biology↗

bioRxiv2023

HLTF Prevents G4 Accumulation and Promotes G4-induced Fork Slowing to Maintain Genome Stability

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes, but their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected, dual role for the dsDNA translocase HLTF in G4 metabolism. First, we find that HLTF is enriched at G4s in the human genome and suppresses G4 accumulation throughout the cell cycle using its ATPase activity. This function of HLTF affects telomere maintenance by restricting alternative lengthening of telomeres, a process stimulated by G4s. We also show that HLTF and MSH2, a mismatch repair factor that binds G4s, act in independent pathways to suppress G4s and to promote resistance to G4 stabilization. In a second, distinct role, HLTF restrains DNA synthesis upon G4 stabilization by suppressing PrimPol-dependent repriming. Together, the dual functions of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.

molecular biology↗

bioRxiv2021

Genome-wide mapping implicates R-loops in lineage-specific processes and formation of DNA break hotspots in neural stem/progenitor cells

Recent work has revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes. However, the mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of DNA:RNA hybrid structures called "R-loops" in primary neural stem/progenitor cells in order to assess their contribution to the different classes of DNA break "hotspots". We report that R-loops in neural stem/progenitor cells are associated primarily with transcribed regions that replicate early and genes that show GC skew in their promoter region. Surprisingly, the majority of genes with recurrent DSB clusters in long, neural genes does not show substantial R-loop accumulation. We implicate R-loops in promoter-proximal DNA break formation in highly transcribed, early replicating regions and conclude that R-loops are not a driver of recurrent double-strand break cluster formation in most long, neural genes. Together, our study provides an understanding of how R-loops may contribute to DNA break hotspots and affect lineage-specific processes in neural stem/progenitor cells.

neuroscience↗

bioRxiv2019

qDRIP: Quantitative differential RNA:DNA hybrid immunoprecipitation sequencing

R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes and cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA:DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here we describe quantitative differential RNA:DNA immunoprecipitation (qDRIP), a method combining synthetic RNA:DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. Finally, we use these quantitative comparisons to make the first estimates of the absolute count of RNA:DNA hybrids per cell and their half-lives genome-wide. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.

molecular biology↗