RNA:DNA hybrids are atypical nucleic acid structures that predominantly form during the transcription of RNA, resulting in the displacement of the non-template strand and the formation of a three-stranded nucleic acid structure, commonly called “R-loop”[1]. However R-loops were also shown to form in trans by invasion of RNA into complementary regions, as demonstrated for telomeres or the pairing of guide RNAs to DNA target sites, a defining feature of type I and type II CRISPR-Cas systems [2], [3], [4], [5], [6]. In addition, Okazaki fragment synthesis during DNA replication leads to RNA:DNA hybrid formation without single-stranded DNA (ssDNA) displacement [7].

R-loops predominantly form behind progressive RNA polymerases (RNPAs), but have also been demonstrated to build up during RNA polymerase backtracking [8]. R-loop levels are dynamic and are modulated by growth conditions and developmental status and have been reported to range from 60 nucleotides to several kilobases. Sequencing-based techniques have revealed that R-loops occupy approximately 5–10 % of the genome, with enrichment at sequences with GC-skew. Consequently, DNA:RNA hybrids frequently promote G-quadruplex (G4) structures in the unpaired ssDNA loop with a corresponding impact on genome stability and gene expression [9], [10], [11], [12], [13], [14], [15].Given the strong link to transcription, R-loops are prevalently genic and were recently subdivided into promoter-paused (Class I) and elongation-associated (Class II) R-loops [16]. R-loops were found to be enriched in proximity to sites of transcriptional initiation, but also at termination sites where RNA:DNA hybrid formation promotes the disengagement of RNA polymerase II (RNAPII) [13], [17]. However, R-loops have also been mapped to enhancers, tRNA genes, rDNA arrays, and repeat-rich regions such as (peri)-centromeres, (sub)-telomeres, and transposable elements [17], [18], [19], [20], [21]. In total, vertebrate cells are estimated to process approximately 27,000 RNA:DNA hybrid structures per day, anticipating the existence of efficient management machineries that keep R-loop levels in check [22], [23], [24], [25].