Chromosomal R-loops: who R they?

R-loops, composed of DNA–RNA hybrids and displaced single-stranded DNA, are known to pose a severe threat to genome integrity. Therefore, extensive research has focused on identifying regulatory proteins involved in controlling R-loop levels. These proteins play critical roles in preventing R-loop accumulation and associated genome instability. Herein I summarize recent knowledge on R-loop regulators affecting R-loop homeostasis, involving a wide array of R-loop screening methods that have enabled their characterization, from forward genetic and siRNA-based screens to proximity labeling and machine learning. These approaches not only deepen our understanding on R-loop formation processes, but also hold promise to find new targets in R-loop dysregulation associated with human pathologies.