R-loops in colorectal cancer: mechanisms, mapping strategies, and therapeutic opportunities.

R-loops are three-stranded nucleic acid structures comprising a DNA: RNA hybrid and a displaced single-stranded DNA strand. Their abnormal persistence can trigger transcription-replication conflicts, replication-fork stalling, DNA damage, chromosomal instability, inflammatory signalling, and epigenetic reprogramming. These effects are particularly relevant to colorectal cancer (CRC), which is frequently characterised by replication stress, defective DNA repair, genomic instability, and treatment resistance. This review summarises current evidence linking altered R-loop homeostasis to CRC initiation, progression, metastasis, molecular heterogeneity, and therapeutic response. We discuss key regulators with evidence in CRC or intestinal models, including RNASEH1/2, DDX21, DHX9, TOP1, POLD1/POLD3, and ATR, while distinguishing them from emerging mechanisms requiring CRC-specific validation. We also evaluate major R-loop detection strategies and their limitations. R-loop-associated vulnerabilities may influence responses to irinotecan, ATR/CHK1 and WEE1 inhibitors, PARP-related strategies, and immunotherapy. However, most evidence remains preclinical, highlighting the need for orthogonal detection methods, patient-derived models, and clinically annotated cohorts.